参见作者原研究论文

本实验方案简略版
May 2020
Advertisement

本文章节


 

A Versatile Protocol to Quantify BCR-mediated Phosphorylation in Human and Murine B Cell Subpopulations
量化人类和小鼠B细胞亚群中BCR介导的磷酸化的通用实验方法   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Signal transduction is the process by which molecular signals are transmitted from the cell surface to its interior, resulting in functional changes inside the cell. B cell receptor (BCR) signaling is of crucial importance for B cells, as it regulates their differentiation, selection, survival, cellular activation and proliferation. Upon BCR engagement by antigen several protein kinases, lipases and linker molecules become phosphorylated. Phosphoflow cytometry (phosphoflow) is a flow cytometry-based method allowing for analysis of protein phosphorylation in single cells. Due to recent advances in methodology and antibody availability – together with the relatively easy quantification of phosphorylation – phosphoflow is increasingly and more commonly used, compared to classical western blot analysis. It can however be challenging to set-up a method that works for all targets of interest. Here, we present a step-by-step phosphoflow protocol allowing the evaluation of the phosphorylation status of signaling molecules in conjunction with extensive staining to identify various human and murine B cell subpopulations, as was previously published in the original paper by Rip et al. (2020). Next to a description of phosphoflow targets from the original paper, we provide directions on additional targets that play a pivotal role in BCR signaling. The step-by-step phosphoflow protocol is user-friendly and provides sensitive detection of phosphorylation of various BCR signaling molecules in human and murine B cell subpopulations.

Keywords: Phosphoflow cytometry (磷酸流式细胞术), Signal transduction (信号转导), B cell receptor signaling (B细胞受体信号转导), B cell subpopulations (B细胞亚群)

Background

B cells are a crucial part of the adaptive immune system and play an important role in protection against pathogens. B cells can sense pathogens by innate receptors such as Toll-like receptors (TLRs). The most important receptor for the development and activation of B cells, however, is the B-cell antigen receptor (BCR). The genes encoding the immunoglobulin heavy and light chains that comprise the BCR are assembled during B cell development in the bone marrow through a stepwise process called V(D)J recombination (Tonegawa, 1983). The functionality and autoreactivity of the BCR are checked during three crucial checkpoints in B cell development: (i) productive heavy chain rearrangement is monitored at the pre-B stage by surface deposition of the heavy chain protein together with pre-existing surrogate light chain as the pre-BCR complex; (ii) productive light chain recombination enables surface expression of the BCR, which is checked for auto-reactivity at the immature B cell stage and (iii) upon bone marrow egress when transitional B cell progress to mature, naive B cells in peripheral lymphoid tissues. When expression of (pre-)BCR signaling molecules is altered, B cell development in the bone marrow is severely affected (Martensson et al., 2010; Pieper et al., 2013). Balanced BCR signaling is also crucial for appropriate selection, survival and activation of mature B cells and its distortion is a key factor in the development of autoimmune disease or leukemia (Nishizumi et al., 1995; Kil et al., 2012; Rip et al., 2018).


Upon BCR engagement by antigen, Src family kinases phosphorylate the intracellular tyrosine-based activation motif (ITAM) of CD79a (Ig-α) and CD79b (Ig-β), which are the transmembrane proteins that form a complex with the BCR, as well the cytoplasmic tail of co-receptor CD19. Phosphorylation of the CD79a/b ITAM in turn activates spleen tyrosine kinase (SYK), which subsequently activates the SH2 domain-containing leukocyte protein of 65 kDa (SLP-65) linker protein. Simultaneously, phosphorylated CD19 will activate phosphoinositide 3-kinase (PI3K), which in turn phosphorylates and converts phosphatidylinositol (4,5)-bisphosphate (PIP2) to phosphatidylinositol (3,4,5)-triphosphate (PIP3). Signaling molecules containing a pleckstrin-homology domain, such as Bruton’s tyrosine kinase (BTK) and phospholipase Cγ2 (PLCγ2), are recruited to the cell membrane by the newly formed PIP3 (Saito et al., 2001). After BTK recruitment to the cell membrane, SLP-65 mediates SYK-dependent activation of BTK, which in turn will phosphorylate PLCγ2 (Wang et al., 2000). The activation of this signaling complex eventually results in activation of protein kinase B (PKB/AKT) signaling (Craxton et al., 1999), NF-κB translocation to the cell nucleus (Bajpai et al., 2000), calcium mobilization (Fluckiger et al., 1998) and activation of the mitogen-activated protein kinase (MAPK) pathway (Jiang et al., 1998). The complex containing BTK, SLP-65, PLCγ2 together with other PI3K-derived signals is essential for B cell survival, proliferation and differentiation. BCR signal transduction is regulated by the equilibrium of kinase activity and negative regulation of signaling through recruitment of tyrosine phosphatases including SHP-1 (Franks and Cambier, 2018).


Western blotting used to be the method of choice for detection and quantification of phosphorylation of the BCR signaling molecules described above. The main drawbacks of Western blotting are the large number of purified cells that are required to perform the analysis and that quantification is not sensitive enough to detect subtle differences between samples. Phosphoflow is a flow-cytometry based technique that allows for quantification of multiple subpopulations within a single sample without prior purification and requires relatively few cells. In addition, phosphoflow cytometry is less time-consuming compared to Western blotting. Although for the quantification of protein phosphorylation phosphoflow appeared to be superior to Western blotting, the fixation and permeabilization procedure of phosphoflow often affected cellar epitopes or features of the monoclonal antibodies (mAbs) that were required to identify the immune cell population of interest. Due to recent advances in phosphoflow procedures, it is now possible to combine the analysis of phosphorylation of signaling proteins with a comprehensive staining for B cell subpopulations. In addition, more and more mAbs become available for phosphoflow analysis.


Here, we present our step-by-step phosphoflow protocol that is complementary to our original research paper (Rip et al., 2020). The phosphoflow protocol allows for sensitive detection of phosphorylation of numerous BCR signaling molecules in human and murine B cell subpopulations. Next to a description of phosphoflow targets as published in the original paper (Rip et al., 2020), we also present additional BCR signaling targets that can be sensitively quantified using this phosphoflow protocol.


Materials and Reagents

Acquisition of human PBMCs from whole blood

  1. Leucosep Tube, 50 ml (Greiner Bio-One, catalog number: 227290 )

  2. 2 ml Cryovial (Starstedt, catalog number: 72.694.006 )

  3. Total peripheral blood (~25 ml) from a healthy donor or patient. In general, 1 ml of blood contains 1 × 106 PBMCs.

  4. Ficoll-PaqueTM Plus (GE Healthcare, catalog number: 17-1440-03 )

  5. Fetal bovine serum (FBS, Gibco, catalog number: 10270-106 )

    CRITICAL: Heat-inactivate for 1 h at 56 °C prior to use.

  6. 20% dimethyl sulfoxide (DMSO, Sigma-Aldrich, catalog number: D2650-100ML)

    CAUTION: DMSO is a toxic product and should be handled with care and discarded.

  7. RPMI 1640 Medium containing L-glutamine (Lonza, catalog number: BE12-702F ) supplemented with 5% (vol/vol) fetal bovine serum (Gibco, catalog number: 10270-106 ) (see Recipes)

  8. Freezing medium containing 80% FBS (Gibco, catalog number: 10270-106 ) (see Recipes)


Collection of Murine organs
  1. 10 ml syringes (Braun, catalog number: 4616103V ) and 25-gauge × ⅝ inch needle (Braun, catalog number: 4657853 )

  2. C57BL/6J mice (Charles River) that are 8-12 weeks of age, either males and females

    Note: All animal experiments should comply with national laws and institutional regulations.

  3. PBS (Gibco, catalog number: 14190169 )


Preparation of Murine organs
  1. 100 µm nylon strainers (Corning, catalog number: 352360 )

  2. 2 ml syringes (BD Biosciences, catalog number: 307727 )

  3. Nunc Cell Culture/Petri dishes (Thermo Scientific, catalog number: 1533066 )

  4. RPMI 1640 Medium, GlutamaxTM Supplement (Gibco, catalog number: 61870036 ) containing 2% (vol/vol) FBS (Capricorn, catalog number: FBS-12A ) (see Recipes)

    CRITICAL: Heat-inactivate for 1 h at 56°C prior to use.


In vitro Stimulation of Single-Cell Suspension/Direct fixation for ex vivo (basal) measurements

  1. 96-wells plate (U-shaped, 310 µl/w, Greiner Bio-One, catalog number: 650201 )

  2. eBioscience FoxP3/Transcription Factor staining kit (Invitrogen, catalog number: 00-5523-00 ) containing:

    1. Fixation and permeabilization concentrate

      CAUTION: This product contains paraformaldehyde (PFA), a toxic and mutagenic product. It should be handled with care and discarded as hazardous waste.

    2. Diluent

    3. 10× Wash Buffer (dilute 10 times in deionized water)

  3. Stimuli for BCR signaling and co-receptor engagement, see Table 1.

    CAUTION: Sodium azide is highly toxic and dangerous to the environment. It should be handled with care and discarded as hazardous waste.


    Table 1. BCR and co-receptor ligands for stimulation of human and murine B cells


Flow Cytometry Procedures

  1. Insert tubes (Greiner Bio-One, catalog number: 102280 ) and 5 ml round bottom polystyrene tubes (Corning, catalog number: 352052 )

  2. UltraComp eBeadsTM Compensation Beads (Invitrogen, catalog number: 01-2222-42 )

  3. 10× Wash Buffer (dilute 10 times in deionized water) from the eBioscience FoxP3/Transcription Factor staining kit (Invitrogen)

  4. FcR Blocking agent (Human TruStain FcX, Biolegend, catalog number: 422302 )

  5. Antibodies for flow cytometry are listed in Table 2 (phospho-protein-specific antibodies) and Table 3 (antibodies used for an accurate identification of B cell subpopulations). For mouse studies, another 2.4G2 antibody was used from own production.

    CAUTION: Sodium azide is highly toxic (see above).

  6. MACS-buffer containing 0.5% (vol/vol) bovine serum albumin (BSA, Sigma-Aldrich, catalog number: A8327 ) and 2 mM ethylenediaminetetraacetic acid solution (EDTA, Sigma-Aldrich, catalog number: 0 3690 ) in PBS (see Recipes)


    Table 2. Phospho-specific antibodies for the analysis of human and mouse B cells and directions on stimulation timing and antibody dilution



    Table 3. Antibodies used for accurate gating of B cell subsets

Equipment

  1. Standard laboratory materials or equipment, such as pipettes, tubes, and tips

  2. Tools suitable for sacrificing mice and harvesting organs, including: scissors, tweezers and 70% ethanol

  3. Mortar and pestle

  4. Stopwatch or timer

  5. Water bath incubator and/or CO2 incubator

  6. Multifuge x 3R Refrigerated Centrifuge (Thermo Scientific, catalog number: 75004515 ) with TX-1000 swinging bucket rotor (Thermo Scientific, catalog number: 75003017 ).

  7. LSR II flow cytometer (BD Biosciences, catalog number: BD LSR-II ) equipped with a red, blue and violet laser

  8. Flow cabinet

  9. 4 °C fridge

  10. -80 °C freezer

Software

  1. Data acquisition by FACSDivaTM Software (BD Biosciences)

  2. FlowJo software (BD Biosciences) to analyze data

Procedure

  1. Sample collection

    Acquisition of human PBMCs from whole blood (Timing: 60 min)

    Note: Steps 1 to 6 are performed in a sterile environment using a flow cabinet.

    1. Add 15 ml of Ficoll to the Leucosep tube and spin down at 1,000 × g for 1 min at room temperature (RT).

    2. Gently load a maximum of 25 ml whole blood per Leucosep tube.

      CRITICAL: Make sure the Ficoll is beneath the filter in the tube and that blood does not mix with the Ficoll.

    3. Spin down the Leucosep tube containing whole blood and Ficoll at 1,000 × g for 10 min at RT.
      CRITICAL: Set the deceleration as low as possible to keep the Ficoll gradient separation of the PBMC layer intact.

    4. Transfer the whole solution of peripheral blood mononuclear cells (PBMCs), Ficoll and Plasma to a 50 ml tube containing 15 ml RPMI 1640 supplemented with 5% FBS.

      CRITICAL: The erythrocyte fraction below the filter should be retained in the Leucosep tube.

    5. Spin down the 50 ml tube at 400 × g for 7 min at 4 °C. Afterwards, remove the supernatant and resuspend in 1-5 ml of cold (4 °C) RPMI-2% FBS.

      OPTIONAL: When performing phosphoflow on fresh PBMCs (non-frozen samples), proceed to Step 9.

    6. Count cells using standard procedures (manual or automated) and store not more than 50 × 106 viable cells per vial by adding Freezing medium in 1:1 ratio to the volume in which the cells are held. Add 0.5 ml of Freezing medium to 0.5 ml of medium containing 20 × 106- 50 × 106 cells. Transfer the cells into 2 ml cryovials that are suitable for freezing and immediately freeze the vials at a rate of 1 °C per minute.

      CRITICAL: As DMSO is harmful to cells, slowly add the freezing medium to the cells in RMPI 5% FBS while gently shaking the tube.

      PAUSE POINT: Cells can be kept at -80 °C for several weeks or in liquid nitrogen for years.
      CAUTION: DMSO is a toxic product and should be handled with care and discarded.

    7. Vials containing frozen PBMCs are thawed using a 37 °C water bath. Upon reaching the liquid phase, cells are immediately transferred into a 15 ml tube followed by slow addition 10 ml of cold (4 °C) RPMI-5% FBS while gently shaking the tube.

      CRITICAL: This transfer should happen immediately when the liquid phase is reached during thawing, as DMSO is harmful to the cells.

    8. Spin down at 400 × g for 7 min at 4 °C. Afterwards, remove the supernatant and resuspend the pellet in 1 ml of cold (4 °C) RPMI 2% FBS.

    9. Count the cells and plate 5 × 105 cells per well in a 96-wells round bottom plate within a volume of 50 μl RPMI 2% FBS for phosphoflow.

      PAUSE POINT: Cells can be kept at 4 °C for 1-2 h before proceeding with the phosphoflow protocol at Step B1.


    Collection of murine organs (Timing: 15 min per mouse)

    1. Mice are sacrificed by cervical dislocation. The protocol allows for other sacrifice methods that do not affect the anatomical region of the organ of interest, including methods that involve anesthetics.

    2. Peritoneal lavage. A midline incision over the abdomen is made to retract the skin, keeping the abdomen intact. A 10 ml syringe, containing 5 ml of ice-cold PBS and 5 ml of air, is attached to the 25-gauge × ⅝ inch needle. The peritoneum is subsequently washed by gently flushing and shaking upon full injection for 1 min. Afterwards, the peritoneal lavage is collected by aspirating the peritoneal cavity with the 10 ml syringe. The lavage is collected in a 15 ml tube and is kept on ice for further processing.

    3. Spleen. The peritoneal cavity can be opened to carefully extract the spleen. The spleen is located at the left flank in the abdomen and can be extracted using tweezers.

    4. Other organs. Next, the Peyer’s patches can be isolated from the small intestine using curved tip tweezers. Lastly, the femur is extracted by two cuts in the pelvic bone and dislocation of the knee. Harvesting of various other anatomical compartments, including bronchoalveolar lavage (BAL), lungs and lymph nodes as previously described (Li et al., 2017). All organs are placed in ice-cold PBS for further processing.


    Preparation of single-cell suspension of various murine organs (Timing: 15 min per mouse)

    1. Spleen tissue is mechanically disrupted using a 100 μm cell strainer on a 50 ml tube and the plunger of a 2 ml syringe. During and following processing of the spleen, strainers are flushed with 5 ml of RPMI 1640 GlutamaxTM supplemented with 2% (vol/vol) FBS (RMPI-2% FBS) to acquire all cells. Peyer’s patches are processed in a similar manner, using a 100 µm cell strainer and a Petri dish. Strainers are flushed with maximal 200 μl of RMPI-2% FBS to acquire all cells.

      CRITICAL: It is important to immediately put the tubes with single cell suspension back on ice or place them in the fridge at 4 °C after processing.

    2. The femur is processed using a mortar and pestle, crushing the bone and acquiring the bone marrow cells in 3 ml of RMPI-2% FBS. The bone marrow cell suspension is transferred through a 100 μm cell strainer on a 50 ml tube to ensure a single cell solution without parts of bone. The bone marrow cell suspension is kept on ice for upcoming procedures.

    3. Peritoneal lavage cells are centrifuged at 400 × g for 7 min at 4 °C and the supernatant is discarded. Peritoneal lavage cell pellets are resuspended in 1 ml of RMPI-2% FBS and are kept on ice for upcoming procedures.

    4. A cell counting sample is taken from all organs to determine the exact number of cells in each sample.

      CRITICAL: To measure ex vivo basal levels of phosphorylation, cells are kept on ice without any stimulation. Immediately proceed to Step C1.

      PAUSE POINT: Cells can be kept at 4 °C for 1-2 h before proceeding with the phosphoflow protocol.


  2. In vitro stimulation of single-cell suspension (Timing: 30 min-6 h)

    1. Stimulation and staining of cells is performed in 96-wells round bottom plates, using 5 × 105 per well (200,000-1,000,000 cells is technically most optimal). 5 × 105 cells per well are transferred in a volume of 50 μl in each well on ice, keeping the cells all time at 4 °C.

    2. For stimulation times of less than 1 h, the 96-wells round bottom plate is placed in a water bath at 37 °C. Make sure only the bottoms of the wells are in contact with the water. For stimulation times longer than 1 h, the stimulus is added while the cells are on ice and subsequently incubated at 37 °C and 5% CO2. The optimal stimulation time for phosphotargets in human and murine samples are stated in the original paper (Rip et al., 2020) and Table 2.

      CRITICAL: Some phospho-targets in murine B cells, mainly downstream signaling molecules as NF-κB and AKT/S6, are receptive to temperature changes and mechanical stress due to isolation of single cells. Rest these samples at 37 °C, preferably in a CO2 incubator, for 3 h prior to stimulation. This is critical for optimal detection and sensitivity of the downstream signaling read-outs. The pre-incubation for these targets on frozen PBMCs is not recommendable, as the viability of these cells is lower compared to fresh material.

      OPTIONAL: For experiments including in vitro treatment of samples with BCR signaling inhibitors, incubate cell suspensions 3 h at 37 °C with BCR signaling for effective pre-treatment. If the signaling target requires resting, pre-treatment can be performed during this resting time.

    3. When stimulating cells in a water bath, wait 1-2 min to let the samples reach 37 °C. Add the stimulating agent by pipetting 50 μl of stimulus to the wells containing 50 μl of sample. The unstimulated control samples are supplemented with 50 μl RMPI-2% FBS as stimulation control. In addition, add 10 µl RPMI-2% FCS containing fixable live/dead viability stain 10 min prior to fixation.

      CRITICAL: As the fixable live/dead viability stain is light sensitive and is not optimal in RPMI medium, prepare this solution freshly and add to the cells within 15 min post preparation.

    4. Dilute the eBioscience FoxP3 fixation and permeabilization buffer 1:1 with the supplied diluent. When the stimulation time is over, fix the cells by adding 100 μl of the diluted fixation and permeabilization buffer for 10 min at 37 °C.

      CAUTION: This product contains paraformaldehyde (PFA), a toxic and mutagenic product. It should be handled with care and discarded as hazardous waste.

      NOTE: The supplied fixation buffer is 4x concentrated and is diluted in the supplied diluent by combining both in equal volume. The diluted fixative solution is added in equal volume the cells to reach the recommended working concentration (50 µl of 4× fixation buffer + 50 µl of diluent to the 100 µl of cells in RPMI).

    5. After 10 min of fixation, centrifuge at 400 × g for 3 min at 4 °C. Carefully remove the supernatant.

    6. Wash by adding 200 µl eBioscience Wash Buffer to all wells and centrifuge at 400 × g for 3 min at 4 °C. Carefully remove the supernatant afterwards. Perform this step two times. Proceed to Step D1.

      CRITICAL: Two times washing is critical in order to remove all fixation and permeabilization buffer.

      PAUSE POINT: Proceed directly to staining for flow cytometry or keep the cells in a volume of 200 µl of eBioscience Wash Buffer at 4 °C. This storage of cells at 4 °C is possible up to 5 days, without impacting the quality of the samples.


  3. Fixation of single-cell suspensions for ex vivo measurements (Timing: 30 min)

    1. Parallel to stimulated cells, 5 × 105 cells per well are transferred in a 96-wells round bottom plate at a volume of 100 µl per well on ice, keeping the cells at 4 °C at all times.

      CRITICAL: This procedure must always be performed at 4 °C, as this is a read-out of ex vivo activation and temperature alterations could be of great influence.

    2. Samples are stained with the fixable live/dead viability stain for 10 min at 4 °C in the dark prior to fixation.

      CRITICAL: As the fixable live/dead viability stain is not optimal in RPMI medium, prepare this solution freshly and add to the cells within 15 min post preparation.

    3. Fix cells with 100 µl of the diluted fixation and permeabilization buffer for 10 min at 4 °C.

    4. After 10 min of fixation, centrifuge at 400 × g for 3 min at 4 °C. Carefully remove the supernatant.

    5. Wash by adding 200 µl eBioscience Wash Buffer to all wells and centrifuge at 400 × g for 3 min at 4 °C. Carefully remove the supernatant afterwards. Perform this step two times.

      CRITICAL: Two times washing is critical in order to remove all fixation and permeabilization buffer.

      PAUSE POINT: Proceed directly to staining for flow cytometry or keep the cells in a volume of 200 μl of eBioscience Wash Buffer at 4 °C. This storage of cells at 4 °C is possible up to 5 days, without impacting the quality of the samples.


  4. Flow cytometry procedures (Timing: 1-2 h)

    1. After the last wash at Step B6 or Step C5, supernatant is removed and the staining mix can be added. Cells are incubated with 40 μl mix to intracellularly stain for cell surface markers such as CD3, B220/CD19, IgD and IgM in eBioscience Wash Buffer. An FcR-blocking agent is added to the staining mix to avoid non-specific binding of the antibodies. Cells are incubated in the dark for 30 min at 4 °C.

      CAUTION: Sodium azide is highly toxic and dangerous to the environment. It should be handled with care and discarded as hazardous waste.

      CRITICAL: Carefully resuspend the pellet in 40 μl mix, ensuring all cells are in suspension.

      NOTE: The staining can also be performed for 15 min at room temperature (RT, ~20 °C) in the dark.

    2. Samples are washed by adding 200 μl eBioscience Wash Buffer to all wells and centrifuge at 400 × g for 3 min at 4 °C. Carefully remove the supernatant afterwards.

    3. Incubate the samples with 40 μl eBioscience Wash Buffer containing the phospho-target antibody of choice for 30 min at RT in the dark.

      OPTIONAL: If the phospho-target antibody is already labeled with a conjugate, proceed to Step D6 upon completion of the incubation time.

    4. Samples are washed by adding 200 μl eBioscience Wash Buffer to all wells and centrifuge at 400 × g for 3 min at 4 °C. Carefully remove the supernatant afterwards.

    5. When the phospho-protein concerns an unconjugated rabbit-derived antibody, incubate with a donkey anti-rabbit PE-conjugated antibody for 15 min at RT in the dark.

    6. Samples are washed with MACS Buffer (0.5% BSA and 2 mM EDTA in PBS) and centrifuged at 400 × g for 3 min at 4 °C. Remove the supernatant and resuspended in 50 μl MACS buffer. Samples are transferred into insert tubes and measured using a flow cytometer within 1 day.

    7. Prior to measuring, calibrate the LSR-II by compensating for all conjugates that are used within the phospho-staining. Preferably use the same antibodies as used in the staining mix and add those to UltraComp eBeads (Invitrogen) according to manufacturer instructions to calibrate the flow cytometer.

    8. Acquire a sufficient number of cells to perform B cell subpopulation analysis. This would imply that acquiring between 20,000-50,000 B cells per sample would be sufficient.

      CRITICAL: Due to the fixation and permeabilization protocol, the cell size will have decreased compared with non-fixed cells. Increase the voltage of the forward-scatter area (FSC-A) accordingly.

    9. Analysis is performed using FlowJo v9 or v10 software.

Data analysis

In this protocol manuscript, we include targets described in the original paper (Rip et al., 2020) and provide directions on the analyses of several additional phospho-targets that we recently established.

    For each target, it is crucial to first find the best stimulus and identify the most optimal time of stimulation. Table 2 provides directions on the optimal stimulation time for BCR signaling molecules mentioned in the original paper (Rip et al., 2020) and additional phospho-targets in mice and men. During optimization of the subset and phospho-target staining procedure, all antibodies should be validated by including isotype and fluorescence minus one (FMO) controls, thereby verifying the signal intensities. Following acquisition of the cells by the flow cytometer, compensation can be checked and adjusted when necessary using the FlowJo 10 Software program.

    As a data analysis example, Figure 1 provides an overview of the phosphorylation status analysis for various BCR signaling molecules in B cells from human PBMC fractions, either unstimulated or stimulated by anti-IgM F(ab')2 fragments or recombinant CD40L. Prior to gating B cell subpopulations, we first selected for live, single lymphocytes followed by the identification of B cells with the pan-B cell marker CD19 (Figure 1A). We included CD3 in the staining to enable accurate gating of B cells as CD19+CD3- cells. Moreover, CD19-CD3+ T cell fractions can serve as a useful negative control for the analysis of phosphorylation of BCR signaling molecules as T cells are not responsive to anti-IgM stimulation. Within live CD19+ B cells, the populations of transitional B cells and plasmablasts were identified as CD27-CD38+ and CD27+CD38+ cells, respectively. B cells with low CD38 expression were subsequently divided into naive CD27-IgD+ B cells and CD27+IgD- memory B cells (Figure 1A).

    As a next step, phosphorylation of BCR signaling molecules was analyzed in gated B cell subpopulations. For example, we gated on CD27-IgD+ naive B cells and analyzed PLCγ2 (Y759) phosphorylation. For this analysis, next to FMO or isotype controls, gated T cells served as a negative staining control, because PLCγ2 is not expressed in T cells. The histogram overlays in Figure 1B show that the pPLCγ2 fluorescence signals of naive B cells were strongly increased upon anti-IgM stimulation, compared with unstimulated naive B cells. It is of note that unstimulated cells often show low but detectable ex vivo phosphorylation of various BCR signaling proteins. Phosphorylation signals were quantified by geometric mean fluorescence intensity (gMFI) values, because we observed stimulation-induced intensity shifts of a single peak in the case of pPLCγ2 (Figure 1B). In contrast, phosphorylation is quantified by calculating proportions of positive cells when a bimodal distribution is observed (for example for phosphorylation of ribosomal protein S6 (Figure 1C). We usually also include a vertical line for reference that is set either (i) at the peak of the counts of the unstimulated control, when stimulation results in an intensity shift of a single peak; or (ii) to indicate background signals, when stimulation increased the numbers of positive cells in a bimodal staining distribution (as in pS6).

    Additional examples include the analysis in total CD19+ B cells fractions of phosphorylation of CD79a and ribosomal protein S6, a downstream target of the AKT signaling pathway (Figure 1C). Finally, we show histogram overlays of phosphorylation targets that were not previously described in Rip et al. (2020), including the activating Y418 (in mouse B cells: Y424) and inhibitory Y507 of Src-family kinases, PI3K p85 subunit (Y458) and the Src homology region 2 domain-containing phosphatase-1 (SHP-1; Y564) (Figure 1D).



Figure 1. Phosphorylation of BCR molecules in PBMC-derived B cells. A. Gating strategy for live single lymphocytes from the peripheral blood mononuclear cell (PBMC) fraction. Within lymphocytes, we gated for CD19+ cells (CD19+CD3-). After exclusion of transitional B cells (Trans) and plasmablasts (PB), we gated for naive B cells (CD19+CD38-IgD+CD27-) and IgD-negative memory B cells (CD19+CD38-IgD-CD27+). B. Histogram overlay for pPLCγ2 in naive B cells after 5 min of anti-IgM stimulation. C. Histogram overlay for pS6 after 30 min of anti-IgM or recombinant human CD40 ligand (CD40L) and IL-4 stimulation, and for pCD79a after 1 min of anti-IgM in total CD19+ B cells. D. Histogram overlays of total CD19+ B cells for pSRC-Y418 after 1 min of anti-IgM stimulation and for pSRC-Y507, pPI3K p85 and pSHP-1 after 3 min of anti-IgM stimulation. Vertical lines in histograms are set on unstimulated controls as reference for stimulation, whereby the quantification by geometric mean fluorescence intensity (gMFI) is indicated. Only for ribosomal protein S6, which showed a bimodal distribution, phosphorylation is quantified by calculating proportions of positive cells (C). Graphs represent representative histograms of 2-10 individual experiments, each with 2-20 healthy donors per group.


    In Figure 2 we present a parallel phospho-analyses on some previously validated (Rip et al., 2020) and novel targets for murine B cells. We selected live splenic B cells by gating on live B220+ cells and subsequently for naive B cells by IgD and IgM receptor expression (Figure 2A). Within naive B cells, we gated on follicular and marginal zone (MZ) B cells, using CD21 and CD23 (Figure 2A). Phosphorylation of PLCγ2 in follicular B cells showed about a 2.5-fold increase upon stimulation with anti-IgM (Figure 2B). Likewise, robust phosphorylation of CD79a following anti-IgM stimulation and of S6 following anti-IgM, anti-CD40 or lipopolysaccharide (LPS) stimulation was detected in naive B cells (Figure 2C). Additional BCR targets also displayed responsiveness following anti-IgM stimulation, with an optimal signal at 5 min for pSrc kinases (for both Y424 and Y507) and 1 min for pPI3K p85 and pSHP-1 (Figure 2D).



Figure 2. Phosphorylation of BCR molecules in splenic murine B cells. A.Gating strategy for live, single splenic lymphocytes. Within lymphocytes, we gated for splenic B220+ cells (B220+CD3-). After purification for naive B cells using IgD and IgM, we gated for marginal zone (MZ) B cells (B220+CD21+CD23-) and follicular (Fol) B cells (B220+CD21-CD23+). B. Histogram overlay for pPLCγ2 after 5 min of anti-IgM stimulation in Fol B cells. C. Histogram overlays for pCD79 after 5 min of anti-IgM stimulation or pS6 after 3 h of anti-IgM, anti-CD40 or Lipopolysaccharide (LPS) stimulation. D. Histogram overlays of total naive B cells for pSrc-Y424 and pSrc-pY507 after 5 min and for pPI3K p85 and SHP-1 after 1 min of anti-IgM stimulation. Vertical lines in histograms are set on unstimulated controls as reference for stimulation, whereby the quantification by geometric mean fluorescence intensity (gMFI) is indicated. Only for ribosomal protein S6, which showed a bimodal distribution, phosphorylation is quantified by calculating proportions of positive cells (C). Graphs represent representative histograms of > 3 individual experiments, each with 5 mice per group; wild-type mice were 8-12 weeks old.

Notes

  1. Timing for phosphorylation of other signaling proteins was presented in our original paper (Figures 1-3 and an schematic overview in Figure S2) (Rip et al., 2020).

  2. It is not required to perform multiple washing steps, since we did not find differences in the quality of the staining between procedures with single or multiple washing steps. Single washing steps are therefore sufficient, unless otherwise indicated within the methods section.

  3. The phosphoflow protocol was developed and validated for RPMI1640 medium. We did not investigate effects of other types of medium or supplements such as HEPES or 2-Mercapto-ethanol.

  4. Dilutions and timing of the previously validated and additional targets shown in the Data analysis are included in Table 2. Note that both mAb dilution and timing for the phospho-antibody can vary between human and murine samples.

  5. Phosphorylation of STAT protein and read-out for the NF-κB signaling pathway should be performed using other protocols, as mentioned in our original paper (Figures 2-3 and Figure S1) (Rip et al., 2020).

  6. In downstream signaling pathways in freshly acquired murine B cells are receptive to temperature changes and mechanical stress due to isolation of single cells. Rest these samples at 37 °C prior to stimulation, preferably in an CO2 incubator. We recommend to rest the samples 3 h, as is critical for optimal detection and sensitivity of the downstream signaling read-outs.

  7. The pre-incubation for downstream signaling is not recommended on frozen PBMCs, as this strongly affects viability. We did not encounter this viability issue on frozen splenic suspensions derived from murine tissue.

Recipes

Solutions are not filtered; media, sera and buffers are sterile, fetal bovine serum (FBS) is heat-inactivated for 1 h at 56 °C prior to use.

  1. Freezing medium

    80% Fetal Bovine Serum

    20% Dimethyl sulfoxide

  2. RPMI-2% FCS

    RMPI 1640

    2% Fetal Bovine Serum

  3. RPMI-5% FBS

    RPMI 1640

    5% Fetal Bovine Serum

  4. MACS Buffer

    Phosphate-buffered saline

    0.5% Bovine Serum Albumin

    2 mM Ethylenediaminetetraacetic acid solution

Acknowledgments

These studies were partially supported by the Dutch Cancer Society (KWF grant 2014-6564) and Target-to-B, both awarded to R.W. Hendriks, as well as an unrestricted grant from AcertaPharma B.V. Oss.

    We would like to thank Allard Kaptein (AcertaPharma B.V., Oss), Stefan Neys and Marjolein de Bruijn (Erasmus MC Rotterdam) and the EDC Erasmus MC animal facility for their great technical assistance.

Author Contributions

JR designed the research, performed experiments, analyzed the data, and wrote the manuscript. OC and RH contributed to the research design and the writing of the manuscript and supervised the study. All co-authors approved the final manuscript.

Competing interests

The authors declare to have no financial conflicts of interest.

Ethics

PBMCs were obtained from healthy volunteers (Rotterdam Centrum, the Netherlands). Experimental procedures were approved by the ethics committee of the Erasmus MC.

    Prior to animal experiments, all experimental protocols were reviewed and approved by the Erasmus MC Committee of animal experiments (DEC) and the central committee for animal experiments (CCD) with approval ID AVD1010020173764. CCD approval is valid until the 1st of June 2023.

References

  1. Bajpai, U. D., Zhang, K., Teutsch, M., Sen, R. and Wortis, H. H. (2000). Bruton's tyrosine kinase links the B cell receptor to nuclear factor kappaB activation. J Exp Med 191(10): 1735-1744.
  2. Craxton, A., Jiang, A., Kurosaki, T. and Clark, E. A. (1999). Syk and Bruton's tyrosine kinase are required for B cell antigen receptor-mediated activation of the kinase Akt. J Biol Chem 274(43): 30644-30650.
  3. Fluckiger, A. C., Li, Z., Kato, R. M., Wahl, M. I., Ochs, H. D., Longnecker, R., Kinet, J. P., Witte, O. N., Scharenberg, A. M. and Rawlings, D. J. (1998). Btk/Tec kinases regulate sustained increases in intracellular Ca2+ following B-cell receptor activation. EMBO J 17(7): 1973-1985.
  4. Franks, S. E. and Cambier, J. C. (2018). Putting on the Brakes: Regulatory Kinases and Phosphatases Maintaining B Cell Anergy. Front Immunol 9: 665.
  5. Jiang, A., Craxton, A., Kurosaki, T. and Clark, E. A. (1998). Different protein tyrosine kinases are required for B cell antigen receptor-mediated activation of extracellular signal-regulated kinase, c-Jun NH2-terminal kinase 1, and p38 mitogen-activated protein kinase. J Exp Med 188(7): 1297-1306.
  6. Kil, L. P., de Bruijn, M. J., van Nimwegen, M., Corneth, O. B., van Hamburg, J. P., Dingjan, G. M., Thaiss, F., Rimmelzwaan, G. F., Elewaut, D., Delsing, D., van Loo, P. F. and Hendriks, R. W. (2012). Btk levels set the threshold for B-cell activation and negative selection of autoreactive B cells in mice. Blood 119(16): 3744-3756.
  7. Li, B. W. S., Beerens, D., Brem, M. D. and Hendriks, R. W. (2017). Characterization of Group 2 Innate Lymphoid Cells in Allergic Airway Inflammation Models in the Mouse. Methods Mol Biol 1559: 169-183.
  8. Martensson, I. L., Almqvist, N., Grimsholm, O. and Bernardi, A. I. (2010). The pre-B cell receptor checkpoint. FEBS Lett 584(12): 2572-2579.
  9. Nishizumi, H., Taniuchi, I., Yamanashi, Y., Kitamura, D., Ilic, D., Mori, S., Watanabe, T. and Yamamoto, T. (1995). Impaired proliferation of peripheral B cells and indication of autoimmune disease in lyn-deficient mice. Immunity 3(5): 549-560.
  10. Pieper, K., Grimbacher, B. and Eibel, H. (2013). B-cell biology and development. J Allergy Clin Immunol 131(4): 959-971.
  11. Rip, J., de Bruijn, M. J. W., Kaptein, A., Hendriks, R. W. and Corneth, O. B. J. (2020). Phosphoflow protocol for signaling studies in human and murine B cell subpopulations. J Immunol 204(10): 2852-2863.
  12. Rip, J., Van Der Ploeg, E. K., Hendriks, R. W. and Corneth, O. B. J. (2018). The role of bruton's tyrosine kinase in immune cell signaling and systemic autoimmunity. Crit Rev Immunol 38(1): 17-62.
  13. Saito, K., Scharenberg, A. M. and Kinet, J. P. (2001). Interaction between the Btk PH domain and phosphatidylinositol-3,4,5-trisphosphate directly regulates Btk. J Biol Chem 276(19): 16201-16206.
  14. Tonegawa, S. (1983). Somatic generation of antibody diversity. Nature 302(5909): 575-581.
  15. Wang, D., Feng, J., Wen, R., Marine, J. C., Sangster, M. Y., Parganas, E., Hoffmeyer, A., Jackson, C. W., Cleveland, J. L., Murray, P. J. and Ihle, J. N. (2000). Phospholipase Cgamma2 is essential in the functions of B cell and several Fc receptors. Immunity 13(1): 25-35.

简介

[背景] 信号转导是分子信号从细胞表面传递到细胞内部的过程,导致细胞内部功能发生变化。B细胞受体(BCR)信号对于B细胞至关重要,因为它调节了它们的分化,选择,存活,细胞活化和增殖。抗原与BCR结合后,一些蛋白激酶,脂肪酶和接头分子会被磷酸化。磷酸流式细胞仪(phosphoflow)是一种基于流式细胞仪的方法,可以分析单个细胞中的蛋白质磷酸化。由于方法学和抗体的可获得性的最新进展,再加上相对容易的磷酸化定量,与传统的蛋白质印迹分析相比,越来越多地使用磷酸流。但是,建立一种适用于所有感兴趣目标的方法可能具有挑战性。在这里,我们提出了逐步的磷流方案,允许对信号分子的磷酸化状态进行评估,并进行广泛的染色,以鉴定各种人和鼠B细胞亚群,如先前在Rip等人的原始论文。,(2020 )。在原始论文中对磷流靶标的描述旁边,我们提供了在BCR信号传导中起关键作用的其他靶标的指导。循序渐进的磷酸流操作流程是用户友好的操作,可对人和鼠B细胞亚群中各种BCR信号分子的磷酸化进行灵敏检测。


[背景] B细胞是适应性免疫系统的关键部分,在预防病原体方面起着重要作用。B细胞可以通过先天性受体(例如Toll样受体)来感知病原体。但是,B细胞发育和激活最重要的受体是B细胞抗原受体(BCR)。编码包含BCR的免疫球蛋白重链和轻链的基因是在骨髓B细胞发育过程中通过称为V(D)J重组的分步过程进行组装的(Tonegawa,1983)。在B细胞发育的三个关键检查点中检查BCR的功能和自身反应性:(i)在B前期通过重链蛋白与先前存在的替代轻链的表面沉积来监测生产性重链重排。 BCR之前的复合体;(ii)生产性轻链重组可实现BCR的表面表达,并在未成熟B细胞阶段检查自身反应性;(iii)当过渡性B细胞发展为外周淋巴组织中成熟的幼稚B细胞时,在骨髓流出时进行检查。当(pre)BCR信号传导分子的表达改变时,骨髓中的B细胞发育受到严重影响(Martensson等,2010; Pieper等,2013)。均衡BCR信号传导也是成熟B细胞的合适的选择,生存和活化关键的和其失真是在自身免疫性疾病或白血病的发展的关键因素(Nishizumi等人,1995;基尔加丹等人,2012;翻录等人。,2018)。

抗原与BCR结合后,Src家族激酶会磷酸化CD79a(Ig-α)和CD79b(Ig-β)的细胞内基于酪氨酸的激活基序(ITAM),它们也是与BCR形成复合物的跨膜蛋白。共同受体CD19的胞质尾巴。CD79a / b ITAM的磷酸化反过来会激活脾酪氨酸激酶(SYK),后者随后会激活65 kD a (SLP-65)接头蛋白的含SH2结构域白细胞蛋白。同时,磷酸化的CD19将激活磷酸肌醇3-激酶(PI3K),进而磷酸化磷脂酰肌醇(4,5)-双磷酸酯(PIP 2 )并将其转化为磷脂酰肌醇(3,4,5)-三磷酸酯(PIP 3 )。含有普列克底物蛋白同源结构域,如布鲁顿信号分子酪氨酸激酶(BTK)和磷脂酶C γ 2(PLC γ 2)中,通过新形成的PIP募集到细胞膜3 (斋藤等人,2001) 。BTK招募后细胞膜,SLP-65介导依赖Syk的BTK,这反过来将磷酸化的PLC激活γ 2(王等人,2000)。在蛋白的激活这个信号复合,最终结果的活化激酶B(PKB / AKT)信令(克拉克斯顿等人,1999),NF- κ B易位至细胞核(巴杰帕伊等人,2000年),钙动员(Fluckiger等,1998)和丝裂原活化的蛋白激酶(MAPK)途径的激活(Jiang等,1998)。复杂的含有BTK,SLP-65,PLC γ 2与其它PI3K衍生的信号加在一起是对于B细胞存活,增殖和分化所必需的。BCR的信号转导受到激酶活性平衡和信号传导负调控的调节,而酪氨酸磷酸酶包括SHP-1 (Franks and Cambier,2018)。

蛋白质印迹曾经是检测和定量上述BCR信号分子磷酸化的一种选择方法。W酯化印迹的主要缺点是进行分析所需的大量纯化细胞,而且定量分析的灵敏度不足以检测样品之间的细微差异。磷酸流是基于流式细胞术的技术,无需事先纯化即可定量单个样品中的多个亚群,并且所需细胞相对较少。此外,流式细胞仪phosphoflow较少相比耗时W¯¯西部时代印迹。尽管对于蛋白质磷酸化的定量,磷酸血流似乎优于W酯印迹法,但是磷酸血流的固定和通透性过程通常会影响细胞表位或单克隆抗体(mAb)的功能,这些功能是鉴定目标免疫细胞群所必需的。由于磷酸流方法的最新进展,现在有可能将信号蛋白的磷酸化分析与对B细胞亚群的全面染色相结合。此外,越来越多的mAb可用于磷流分析。

在这里,我们介绍了逐步的磷流方案,该方案是对我们原始研究论文的补充(Rip等,2020)。phosphoflow协议允许敏感地检测人和鼠B细胞亚群中许多BCR信号分子的磷酸化。在原始论文(Rip等人,2020)发表的磷酸流靶标描述旁边,我们还提出了其他BCR信号传导靶标,可以使用这种磷酸流仪方案对它们进行灵敏地定量。

关键字:磷酸流式细胞术, 信号转导, B细胞受体信号转导, B细胞亚群

材料和试剂
从全血中获取人PBMC
Leucosep管,50米升(格雷纳生物ø NE,目录号:227290)
2毫升Cryovial(Starstedt,目录号:72.694.006)
来自健康供体或患者的总外周血(〜25 ml)。通常,每1毫升血液中含有1 × 10 6个PBMC。
Ficoll-Paque TM Plus(GE H ealthcare,目录号:17-1440-03)
胎牛血清(FBS,Gibco,目录号:10270-106)
关键:使用前在56 °C加热灭活1小时。

20%二甲基亚砜(DMSO,Sigma - Aldrich,目录号:D2650-100ML)
注意:DMSO是有毒产品,应小心处理并丢弃。

含L-谷氨酰胺(Lonza,目录编号:BE12-702F)的RPMI 1640培养基,添加5%(vol / vol)胎牛血清(Gibco,目录编号:10270-106)(请参见食谱)
含80%FBS的冷冻培养基(Gibco,目录号:10270-106)(请参阅食谱)

小鼠器官的收集

10毫升注射器(布劳恩,目录号:4616103V)和25号× ⅝英寸针器(Braun,目录号:4657853)
雄性和雌性8-12周龄的C57BL / 6J小鼠(查尔斯河)
注意:所有动物实验均应遵守国家法律和机构规定。

PBS(Gibco,目录号:14190169)

小鼠器官的制备

100 µm尼龙滤网(Corning,目录号:352360)
2 ml注射器(BD Biosciences,目录号:307727)
Nunc细胞培养/培养皿(Thermo Scientific ,目录号:1533066)
RPMI 1640培养基,Glutamax TM补充剂(Gibco,目录号:61870036)包含2%(vol / vol)FBS(摩Cap座,目录号:FBS-12A)(请参阅食谱)
关键:使用前在56 °C加热灭活1小时。

单细胞悬浮/直接固定的体外刺激,用于离体(基础)测量

96孔板(U型,310微升/瓦特,格雷纳生物ö NE,目录号:650201)
eBioscience FoxP3 /转录因子染色试剂盒(Invitrogen,目录号:00-5523-00),其中包含:
固定和通透浓缩物
注意:本产品包含有毒和致突变性的多聚甲醛(PFA)。应小心处理,并作为危险废物丢弃。

冲淡
10 ×洗涤缓冲液(在去离子水中稀释10倍)
BCR信号传导和共受体参与的刺激,请参见表1 。
注意:叠氮化钠有剧毒,对环境具有危险。应小心处理,并作为危险废物丢弃。


表1.刺激人和鼠B细胞的BCR和共受体配体

刺激物

工作浓度

制造商

猫。没有。


人的



山羊F(ab')2抗人IgM-UNLB

20微克/立方米升

南方生物技术

2022-01

重组人CD40L

2微克/立方米升

研发部

6245-CL / CF

重组人IL-4蛋白

250毫微克/米升

研发部

204-IL-CF






AffiniPure F(ab')2片段山羊抗小鼠IgM

25 μ克/米升

杰克逊免疫研究

115-006-075

纯化的大鼠抗小鼠CD40(克隆3/23)

4 μ克/米升

BD生物科学

553787

脂多糖(LPS)E. Ç OLI 026:B6

400毫微克/米升

西格玛-奥德里奇

L8274


流式细胞仪程序

插入管(格雷纳生物ø NE,目录号:102280)和5米升圆底聚苯乙烯试管中(Corning,目录号:352052)
UltraComp eBeads TM补偿珠(Invitrogen,目录号:01-2222-42)
来自eBioscience FoxP3 /转录因子染色试剂盒(Invitrogen)的10 ×洗涤缓冲液(在去离子水中稀释10倍)
FcR阻断剂(Human TruStain FcX,Biolegend,目录号:422302)
表2(磷酸化蛋白特异性抗体)和表3(用于准确鉴定B细胞亚群的抗体)中列出了用于流式细胞术的抗体。对于小鼠的研究,另一个2.4G2抗体是利用d自己生产。
注意:叠氮化钠是剧毒的(如上所述)。

含有0.5%(vol / vol)牛血清白蛋白(BSA,Sigma-Aldrich,目录号:A8327)和2 mM乙二胺四乙酸溶液(EDTA,Sigma-Aldrich,目录号:03690)的MACS缓冲液(参见配方)

表2.用于分析人和小鼠B细胞的磷酸化特异性抗体以及刺激时机和抗体稀释的方向

抗体

共轭

克隆

制造商

猫。没有。

检测人

单克隆抗体稀释人

检测鼠标

mAb稀释鼠

pAKT(S473)

--

D9E

细胞信号技术

4060升

30-60分钟

100倍

180分钟

100倍

pAKT(T308)

--

D25E6

细胞信号技术

13038升

30-60分钟

50倍

180分钟

100倍

pBTK / pITK(Y223 / Y180)

Alexa Fluor 647

N35-86

蓝光

564846

5分钟

3000倍

5分钟

3000倍

pCD79a(Y182)

Alexa Fluor 647

D1B9

细胞信号技术

29742S

1分钟

50倍

5分钟

50倍

pERK1 / 2(T202 / Y204)

聚乙烯

20A

蓝光

561991

5分钟

8倍

5分钟

8倍

pPI3K p85(Y458 )

--

E3UI1H

细胞信号技术

17366S

1分钟

100倍

3分钟

200倍

PPLC γ 2(Y759)

Alexa Fluor 647

K86-689.37

蓝光

558498

5分钟

8倍

5分钟

8倍

pS6(S240 / 244)

--

D68F8

细胞信号技术

5364

30-60分钟

200倍

180分钟

200倍


pSHP-1(Y564)

--

D11G5

细胞信号技术

8849S

1分钟

100倍

3分钟

500倍

pSLP-65(Y84)

聚乙烯

J117-1278

蓝光

558442

2分钟

8倍

5分钟

8倍

pSrc(Y418 / Y424)

--

EP503Y

Abcam

Ab40660

1-3分钟

200倍

5分钟

500倍

pSrc(Y507)

--

5B6

Abwiz Bio

2461S

1-3分钟

500倍

5分钟

1500倍

pSYK(348)

聚乙烯

I120-722

蓝光

558529

2分钟

8倍

5分钟

8倍




表3.用于精确控制B细胞亚群的抗体

抗体

共轭

克隆

制造商

猫。没有。

单克隆抗体稀释

人的





CD3

Alexa Fluor 700

UCHT1

英杰

56-0038-42

200倍

CD19

FITC

HIB19

BD生物科学

555412

15倍

CD27

BV421

M-T271

BD生物科学

562513

10倍

CD38

BV785

HIT2

生物传奇

303530

15倍

抗体

BV605

IA6-2

BD生物科学

563313

400倍







B220

AF700

RA3-6B2

英杰

56-0452-82

200倍

CD21 / CD35

FITC

eBio4E3

英杰

11-0212-85

100倍

CD23

生物素

B3B4

BD生物科学

553137

400倍

CD3

BV421

145-2C11

BD生物科学

562600

400倍

抗体

BV711

11-26c.2a

BD生物科学

564275

2000倍

免疫球蛋白

PE-Cy7

II / 41

BD生物科学

25-5790-82

400倍

人与鼠





固定染料

eFluor 506



英杰

65-0866-14

200倍

链霉亲和素

APC-eFluor 780



英杰

47-4317-82

200倍

链霉亲和素

BV786



BD生物科学

563858

200倍



版权所有©20 21作者;保留所有权利。独家被许可人生物协议有限责任公司。1个                                                                                                                           


说明:logonew               
设备


标准实验室材料或设备,例如移液器,试管和吸头
适用于牺牲小鼠和采集器官的工具,包括:剪刀,镊子和70%的乙醇
研钵和研杵
秒表或计时器
水浴培养箱和/或CO 2培养箱
具有TX-1000旋斗转子(Thermo Scientific,目录号:75003017)的Multifuge x 3R冷冻离心机(Thermo Scientific,目录号:75004515)。
LSR II流式细胞仪(BD Biosciences,目录号:BD LSR-II),配有红色,蓝色和紫色激光
流量柜
4℃ ˚F脊
-80 °C冷冻室

软件


通过FACSDiva TM软件(BD Biosciences)获取数据
FlowJo软件(BD Biosciences)来分析数据

程序


样品采集
从全血中获取人PBMC(时间:60分钟)

Ñ OTE :步骤PS 1〜6中使用流柜的无菌环境中进行。

向Leucosep管中加入15 ml的Ficoll,然后在室温(RT)下以1,000 × g的转速旋转1分钟。
每个Leucosep管轻柔地加入最多25 ml全血。              
严重:确保Ficoll处于试管中的过滤器下方,并且血液不会与Ficoll混合。

在室温下,以1,000 × g的速度旋转含有全血和Ficoll的Leucosep管,持续10分钟。关键:将减速度设置得尽可能低,以保持PBMC层的Ficoll梯度分离完好无损。
将外周血单核细胞(PBMC),Ficoll和血浆的全部溶液转移到装有15 ml RPMI 1640的5%FBS的50 ml管中。              
关键:过滤器下方的红细胞部分应保留在Leucosep管中。

在4下以500 × g的转速旋转50 ml试管7分钟 ℃。然后,除去上清液,并重悬于1-5 ml的冷(4 °C)RPMI-2%FBS中。
可选:对新鲜的PBMC(非冷冻样品)进行磷流处理时,请继续执行步骤9 。

使用标准程序(手动或自动)对细胞进行计数,并通过将冷冻培养基以1:1的比例添加到保存细胞的体积中,每个小瓶不超过50 × 10 6个活细胞。将0.5ml冷冻培养基的0.5毫升含培养基的20 × 10 6 - 50 × 10 6细胞。将细胞转移到适合冷冻的2 ml冷冻管中,并以每分钟1 °C的速度立即冷冻小瓶。
关键:由于DMSO对细胞有害,因此在摇动试管的同时,将冷冻培养基缓慢加入5%FPI的RMPI细胞中。           
暂停点:细胞可以在-80 °C下保持数周,或在液氮中保持数年。注意:DMSO是有毒产品,应小心处理并丢弃。

使用37 °C水浴将含有冷冻PBMC的样品瓶解冻。达到液相后,立即将细胞转移到15 ml试管中,然后缓慢加入10 ml冷(4 °C)RPMI-5%FBS,同时轻轻摇动试管。
关键:解冻过程中达到液相时应立即发生这种转移,因为DMSO对细胞有害。

在4 °C下以400 × g旋转7分钟。然后,除去上清液,然后将沉淀重悬于1 ml的冷(4 °C)RPMI 2%FBS中。
计数细胞,和板5 × 10 5的50的体积内每孔的细胞在96孔圆底板微升为phosphoflow RPMI 2%FBS。
暂停点:在进行步骤B1的磷酸流操作之前,可以将细胞在4 °C下保持1-2 h 。


收集小鼠器官(时间:每只小鼠15分钟)

颈脱位法处死小鼠。该协议允许其他不影响目标器官解剖区域的牺牲方法,包括涉及麻醉剂的方法。
腹膜灌洗。在腹部上做一个中线切口,以使皮肤缩回,保持腹部完整。的10ml注射器,含有5ml冰冷的PBS和5ml空气,被附接到25号× ⅝英寸针头。腹膜随后通过洗涤gentl ÿ冲洗并在完全注射1分钟摇动。之后,通过用10 ml注射器抽吸腹腔收集腹腔灌洗液。灌洗液收集在15毫升试管中,并置于冰上进行进一步处理。
脾。可以打开腹膜腔以小心地取出脾脏。脾脏位于腹部的左侧腹,可以用镊子拔出。
其他器官。Ñ分机,淋巴集结可以使用弯曲顶端镊子小肠中分离。最后,通过两次切开骨盆骨和使膝盖脱位来抽出股骨。先前已描述了其他各种解剖隔室的收获,包括支气管肺泡灌洗(BAL),肺和淋巴结肿大(Li等,2017)。将所有器官置于冰冷的PBS中进行进一步处理。

制备各种鼠类器官的单细胞悬液(时间:每只小鼠15分钟)

使用50 ml管上的100μm细胞过滤器和2 ml注射器的柱塞机械破坏脾脏组织。在脾脏处理过程中和之后,用5 ml RPMI 1640 Glutamax TM冲洗过滤器,并补充2%(vol / vol)FBS(RMPI-2%FBS)以获取所有细胞。淋巴集结以类似的方式进行处理,使用100 μ米细胞过滤器和皮氏培养皿。用最大200μl的RMPI-2%FBS冲洗过滤器,以获取所有细胞。
CRITICAL:立即把管在冰上或代替它们在冰箱在4单细胞悬浮液回是很重要℃下处理之后。

用研钵和研棒处理股骨,压碎骨头,并在3 ml RMPI-2%FBS中获得骨髓细胞。骨髓细胞悬液通过50毫升试管上的100微米细胞过滤器转移,以确保没有骨部分的单细胞溶液。将骨髓细胞悬液保存在冰上,以备后用。
将腹膜灌洗细胞在4 °C下以400 × g离心7分钟,并弃去上清液。将腹膜灌洗细胞沉淀重悬于1 ml的RMPI-2%FBS中,并保存在冰上以备后用。              
从所有器官中采集细胞计数样品,以确定每个样品中的确切细胞数量。             
至关重要:要测量离体的基础磷酸化水平,将细胞保持在冰上,无需任何刺激。立即进入步骤C1 。

暂停点:在进行磷流方案之前,可以将细胞在4 °C保持1-2小时。


体外单细胞悬液的刺激(时间:30分钟- 6小时)
刺激和细胞的染色在96孔圆底平板中进行,使用5- × 10 5每孔(200,000 - 1,000,000个细胞在技术上是最优化的)。5 × 10 5细胞每孔中50的体积被转移μ升每孔在冰上,保持单元中的所有时间,在4 ℃下。
如果刺激时间少于1小时,则将96孔圆形底板置于37 °C的水浴中。确保仅孔的底部与水接触。对于长于1 h的刺激时间,在细胞在冰上的同时添加刺激,然后在37 °C和5%CO 2下孵育。原始论文(Rip et al。,2020)和表2列出了人类和鼠类样品中磷酸靶的最佳刺激时间。             
关键:鼠B细胞中的一些磷酸化靶标,主要是下游信号分子,如NF-κB和AKT / S6,可以接受由于单细胞分离而产生的温度变化和机械应力。在刺激之前,将这些样品在37 °C下放置3 h ,最好在CO 2培养箱中放置3 h。这对于优化检测和下游信号读出的灵敏度至关重要。不建议在冷冻的PBMC上对这些靶标进行预孵育,因为与新鲜材料相比,这些细胞的活力较低。

可选:对于包括用BCR信号抑制剂体外处理样品的实验,将细胞悬浮液与BCR信号在37 °C下孵育3小时,以进行有效的预处理。如果发信号目标需要休息,则可以在此休息时间内进行预处理。

在水浴中刺激细胞时,请等待1-2分钟,使样品达到37 °C 。吹打50添加刺激剂μ升刺激到含50孔μ升样品。未刺激的对照样品补充有50 μ升RMPI-2%FBS作为刺激控制。此外,添加10 μ升RPMI-2%FCS包含可固定的活/死染色存活固定之前10分钟。                            
关键:由于可固定的活/死活力染色剂对光敏感,在RPMI培养基中并非最佳,因此应重新制备此溶液,并在制备后15分钟内添加到细胞中。

用随附的稀释剂将eBioscience FoxP3固定液和通透缓冲液1:1稀释。当刺激时间结束时,加入100固定细胞μ升在37稀释的固定和透化缓冲液中10分钟℃下。
注意:本产品包含有毒和致突变性的多聚甲醛(PFA)。应小心处理,并作为危险废物丢弃。           
注意:随附的固定缓冲液浓度为4倍,并通过等体积混合在稀释液中稀释。将稀释的固定液按等体积加入细胞中,以达到推荐的工作浓度(在RPMI中的100 µl细胞中加入50 µl 4 ×固定缓冲液+ 50 µl稀释剂)。

固定10分钟后,在4 °C下以400 × g离心3分钟。小心除去上清液。
在所有孔中加入200 µl eBioscience洗涤缓冲液进行洗涤,并在4 °C下以400 × g离心3分钟。然后小心除去上清液。执行两次此步骤。进行步骤D1 。              
关键:两次清洗对于去除所有固定液和通透性缓冲液至关重要。

暂停点:直接进行染色以进行流式细胞术或将细胞保持在4 °C的200 µl eBioscience Wash Buffer体积中。这种细胞在4 °C下最多可以保存5天,而不会影响样品的质量。


固定单细胞悬液以进行离体测量(时间:30分钟)
与受刺激的细胞平行,将每孔5 × 10 5个细胞转移到96孔圆形底板中,每孔100μl的体积放在冰上,使细胞始终保持在4 °C 。              
批判:该程序必须始终在4 °C下进行,因为这是对离体激活的一种读出,温度变化的影响可能很大。

在固定之前,将样品在黑暗中于4 °C下用可固定的活/死生存力染色剂染色10分钟。             
关键:由于在RPMI培养基中固定的活/死活力染色并非最佳,因此应重新制备此溶液,并在制备后15分钟内添加到细胞中。

用100 µl稀释的固定液和通透缓冲液在4 °C下固定细胞10分钟。
固定10分钟后,在4 °C下以400 × g离心3分钟。小心除去上清液。
在所有孔中加入200 µl eBioscience洗涤缓冲液进行洗涤,并在4 °C下以400 × g离心3分钟。然后小心除去上清液。执行两次此步骤。
关键:两次清洗对于去除所有固定液和透化缓冲液至关重要。

暂停点:直接前进到染色用于流式细胞术或使细胞保持在200的体积微升eBioscience的洗涤缓冲液,在4 ℃下。这种细胞在4 °C下最多可以保存5天,而不会影响样品的质量。


流式细胞仪程序(时间:1-2小时)
在步骤B6或步骤C5进行最后一次清洗后,去除上清液,并可以添加染色混合物。细胞与40 μ升组合,以用于细胞表面标记,如CD3,B220 / CD19,IgD和IgM抗体eBioscience的洗涤缓冲液胞内染色。将FcR阻断剂添加到染色混合物中,以避免抗体的非特异性结合。将细胞在黑暗中于4 °C孵育30分钟。
注意:叠氮化钠有剧毒,对环境有害。应小心处理,并作为危险废物丢弃。

关键:小心地将沉淀重悬于40μl混合物中,确保所有细胞均处于悬浮状态。

注意:也可以在黑暗中于室温(RT,〜20 °C )下进行15分钟的染色。

样品通过加入200洗涤微升在400 eBioscience的洗涤缓冲液到所有孔中,并离心×克在4 3分钟℃下。然后小心除去上清液。
将样品与40μl含所选磷酸目标抗体的eBioscience Wash Buffer在黑暗中于室温孵育30分钟。
可选:如果磷酸化目标抗体已被缀合物标记,则在孵育时间结束后继续执行步骤D6。

样品通过加入200洗涤微升在400 eBioscience的洗涤缓冲液到所有孔中,并离心×克在4 3分钟℃下。然后小心除去上清液。
当磷蛋白涉及未结合的兔衍生抗体时,与驴抗兔PE结合抗体在黑暗中于室温孵育15分钟。
样品用MACS缓冲液(PBS中的0.5%BSA和2 mM EDTA)洗涤,并在4 °C下以400 × g离心3分钟。除去上清液并重新悬浮于50微升MACS缓冲液。将样品转移到插入管中,并在1天内使用流式细胞仪进行测量。
在测量之前,通过补偿在磷酸染色中使用的所有结合物来校准LSR-II。优选使用与染色混合物中使用的抗体相同的抗体,并根据制造商的说明将其添加到UltraComp eBeads(Invitrogen)中,以校准流式细胞仪。
获取足够数量的细胞以执行B细胞亚群分析。这意味着每个样品采集20,000-50,000个B细胞就足够了。
关键:由于固定和通透性方案,与非固定细胞相比,细胞大小将减小。相应地增加前向散射区(FSC-A)的电压。

使用FlowJo v9或v10软件执行分析。

数据分析


在该协议手稿中,我们包括原始论文中描述的靶标(Rip等,2020),并提供了对我们最近建立的其他几个磷酸化靶标进行分析的指导。

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            对于每个目标,至关重要的是首先找到最佳刺激并确定最佳刺激时间。表2提供了有关原始论文中提到的BCR信号分子的最佳刺激时间的指导(Rip等,2020),以及小鼠和男性中的其他磷酸化靶标。在优化子集和磷酸化目标染色过程中,应通过包括同种型和荧光减一(FMO)对照来验证所有抗体,从而验证信号强度。用流式细胞仪采集细胞后,可以在必要时使用FlowJo 10软件程序检查和调整补偿。           
                                                          作为数据分析示例,图1概述了未受抗IgM F(ab')2片段或重组CD40L刺激或刺激的人PBMC组分在B细胞中各种BCR信号分子的磷酸化状态分析。在对B细胞亚群进行门控之前,我们首先选择了活的单个淋巴细胞,然后用pan-B细胞标记CD19鉴定B细胞(图1A)。我们在染色包括CD3,使B细胞CD19的精确门+ CD3 -细胞。此外,由于T细胞对抗IgM刺激无反应,因此CD19 - CD3 + T细胞级分可以用作BCR信号分子磷酸化分析的有用阴性对照。在活的CD19 + B细胞中,过渡性B细胞和成浆细胞的群体分别被鉴定为CD27 - CD38 +和CD27 + CD38 +细胞。B细胞与低CD38表达随后被分成娜我已经CD27 - IGD + B细胞和CD27 + IGD -记忆B细胞(图1A )。

              下一步,在门控B细胞亚群中分析BCR信号分子的磷酸化。例如,我们筛选了CD27 - IgD +幼稚B细胞,并分析了PLCγ2(Y759)的磷酸化。对于该分析,毗邻FMO或同种型对照,门控的T细胞用作阴性染色对照,因为PLC γ 2没有在T细胞中表达。直方图overla在图1B YS表明,在p幼稚B细胞的PLCγ2荧光信号在抗IgM刺激强烈增加,未刺激相比NA我已经B细胞。值得注意的是,未刺激的细胞通常显示出低水平但可检测到的各种BCR信号蛋白的离体磷酸化。磷酸化信号通过几何平均荧光强度(g MFI)值进行量化,因为在pPLCγ2的情况下,我们观察到了刺激诱导的单个峰强度变化(图1B)。相反,当观察到双峰分布时,磷酸化通过计算阳性细胞的比例来量化(例如,核糖体蛋白S6的磷酸化(图1C)。我们通常还包括一条垂直线作为参考,该垂直线设置为(i)在当刺激导致单个峰的强度移动时,未刺激对照计数的最大峰值;或(ii)指示背景信号,当刺激增加双峰染色分布中阳性细胞的数量时(如pS6)。

              其他示例包括分析CD79a和核糖体蛋白S6(AKT信号通路的下游靶标)的总CD19 + B细胞的磷酸化水平(图1C )。最后,我们显示了磷酸化目标的直方图叠加图,Rip等人以前没有对此进行过描述。(2020 ),包括激活的Y418(在小鼠B细胞中为Y424)和对Src家族激酶,PI3K p85亚基(Y458)和具有Src同源区域2域的磷酸酶-1(SHP-1; Y564)的抑制性Y507。 (图1D)。





图1. PBMC衍生的B细胞中BCR分子的磷酸化。A.外周血单核细胞(PBMC)部分中活单淋巴细胞的门控策略。在淋巴细胞,我们门控CD19 +细胞(CD19 + CD3 - )。过渡B细胞(反式)和浆(PB)的排除后,我们门控幼稚B细胞(CD19 + CD38 - IGD + CD27 - )和IgD阴性的记忆B细胞(CD19 + CD38 - IGD - CD27 + )。乙。抗IgM刺激5分钟后,幼稚B细胞中pPLCγ2的直方图叠加图。Ç 。抗IgM或重组人CD40配体(CD40L)和IL-4刺激30分钟后pS6的直方图叠加图,总CD19 + B细胞中抗IgM 1分钟后的pCD79a的直方图叠加图。d 。抗IgM刺激1分钟后的pSRC-Y418和抗IgM刺激3分钟后的pSRC-Y507,pPI3K p85和pSHP-1的总CD19 + B细胞的直方图叠加图。将直方图中的竖线设置在未刺激的控件上作为刺激的参考,从而指示通过几何平均荧光强度(g MFI)进行的定量。仅对于显示双峰分布的核糖体蛋白S6,通过计算阳性细胞的比例(C)定量磷酸化。图代表2-10个独立实验的代表性直方图,每组每个实验有2-20个健康供体。           

              在图2中,我们提供了一些先前经过验证的平行磷酸分析(Rip等,2020)和鼠B细胞的新型靶标。我们通过门控活B220选择活脾B细胞+细胞,并随后为NA我已经通过IgD和IgM的雷杰普B细胞TOR表达(图2A )。内娜我已经B细胞,我们选通滤泡和边缘区(MZ)B细胞,使用CD21和CD23(图2A )。抗IgM刺激后,卵泡B细胞中PLCγ2的磷酸化增加了约2.5倍(图2B )。同样地,以下的CD79A健壮磷酸化抗IgM刺激和S6的下列抗IgM,抗CD40或脂多糖(LPS)刺激检测NA我已经B细胞(图2C )。在抗IgM刺激后,另外的BCR靶标也显示了响应性,对于pSrc激酶(对于Y424和Y507而言)在5分钟时,对于pPI3K p85和pSHP-1在1分钟时具有最佳信号(图2D )。           
           



图2.脾脏鼠B细胞中BCR分子的磷酸化。A.活的单个脾淋巴细胞的门控策略。在淋巴细胞,我们选通脾B220 +细胞(B220 + CD3 - )。纯化后为NA我已经使用IgD和IgM的B细胞,我们门控边缘区(MZ)的B细胞(B220 + CD21 + CD23 - )和滤泡(FOL)的B细胞(B220 + CD21-CD23 + )。B. Fol B细胞中抗IgM刺激5分钟后pPLCγ2的直方图叠加图。C.抗IgM刺激5分钟后pCD79的直方图叠加图或抗IgM,抗CD40或脂多糖(LPS)刺激3小时后pS6的直方图叠加图。D.抗IgM刺激5分钟后,pSrc-Y424和pSrc-pY507以及pPI3K p85和SHP-1的总幼稚B细胞的直方图叠加图。将直方图中的竖线设置在未刺激的控件上作为刺激的参考,从而指示通过几何平均荧光强度(g MFI)进行的定量。仅对于显示双峰分布的核糖体蛋白S6,通过计算阳性细胞的比例(C)定量磷酸化。图代表> 3个独立实验的代表性直方图,每组各有5只小鼠;野生型小鼠为8-12周大。           

笔记


定时为其他信令蛋白的磷酸化是在我们的最初的纸(图1-3和图S2的示意性概述)呈现(翻录等人,2020) 。
不需要执行多个洗涤步骤,因为在单个或多个洗涤步骤的步骤之间我们没有发现染色质量的差异。因此,除非方法部分中另有说明,否则单步洗涤就足够了。
针对RPMI1640培养基开发并验证了phosphorflow方案。我们没有研究其他类型的培养基或补充剂(例如HEPES或2-巯基乙醇)的作用。
的在数据中示出的先前验证和额外的目标稀释和定时一个nalysis被包括在表2中。请注意,人和鼠样品之间的mAb稀释和磷酸化抗体的时间安排可能会有所不同。
STAT蛋白和读出为磷酸化的NF- κ乙小号应该使用其它协议如在我们的最初的纸(图中提到执行ignaling途径,小号2-3和图S1) (翻录等人,2020) 。
在新鲜获得的鼠B细胞的下游信号传导途径中,由于单个细胞的分离,它们可以接受温度变化和机械应力。刺激前,最好将其置于37 °C的CO 2培养箱中。我们建议将样品静置3 h,因为这对于优化检测和下游信号读数的灵敏度至关重要。
不建议在冷冻的PBMC上预孵育下游信号,因为这会严重影响生存能力。在衍生自鼠类组织的冷冻脾悬液中,我们没有遇到这种生存能力的问题。

菜谱


解决方案未过滤;培养基,血清和缓冲液是无菌的,使用前将胎牛血清(FBS)在56 °C加热灭活1小时。

冷冻介质
80%胎牛血清

20%二甲基亚砜

RPMI-2%FCS
RMPI 1640

2%胎牛血清

RPMI-5%FBS
RPMI 1640

5%胎牛血清

MACS缓冲区
磷酸盐缓冲溶液

0.5%牛血清白蛋白

2 mM乙二胺四乙酸溶液


致谢


这些研究得到了荷兰癌症协会(KWF赠款2014-6564)和Target-to-B的部分支持,两者均授予RW Hendriks,以及AcertaPharma BV Oss的无限制赠款。

  我们要感谢Allard Kaptein(AcertaPharma BV,奥斯),Stefan Neys和Marjolein de Bruijn(伊拉斯姆斯MC鹿特丹)以及EDC伊拉斯姆斯MC动物设施提供的巨大技术援助。           

作者贡献

  JR设计了研究,进行了实验,分析了数据并撰写了手稿。OC和RH为研究设计和手稿的撰写做出了贡献,并对研究进行了监督。所有合著者都批准了最终稿。


竞争我nterests


作者宣称没有经济利益冲突。


伦理


PBMC来自健康志愿者(荷兰鹿特丹中心)。实验程序已由Erasmus MC的道德委员会批准。           
  在进行动物实验之前,所有实验方案均由Erasmus MC动物实验委员会(DEC)和动物实验中央委员会(CCD)审查并批准,批准ID为AVD1010020173764 。CCD的批准有效期至2023年6月1日。



参考文献


Bajpai,UD,Zhang,K.,Teutsch,M.,Sen,R。和Wortis,HH(2000)。布鲁顿的酪氨酸激酶将B细胞受体与核因子kappaB激活联系起来。J Exp Med 191(10):1735-1744。
Craxton,A.,Jiang,A.,Kurosaki,T.和Clark,EA(1999)。B细胞抗原受体介导的激酶Akt激活需要Syk和Bruton的酪氨酸激酶。生物化学杂志274(43):30644-30650。
Fluckiger,AC,Li,Z.,Kato,RM,Wahl,MI,Ochs,HD,Longnecker,R.,Kinet,JP,Witte,ON,Scharenberg,AM和Rawlings,DJ(1998)。Btk / Tec激酶调节B细胞受体激活后细胞内Ca 2+的持续增加。EMBO J 17(7):1973-1985。
SE的Franks和JC的Cambier(2018)。P的Utting刹车:法规激酶和磷酸酶维护乙细胞无反应性。 免疫前线9:665。
Jiang,A.,Craxton,A.,Kurosaki,T.和Clark,EA(1998)。B细胞抗原受体介导的细胞外信号调节激酶,c-Jun NH2末端激酶1和p38丝裂原活化蛋白激酶的激活需要不同的蛋白质酪氨酸激酶。实验医学杂志188(7):1297-1306。
Kil,LP,de Bruijn,MJ,van Nimwegen,M.,Corneth,OB,van Hamburg,JP,Dingjan,GM,Thaiss,F.,Rimmelzwaan,GF,Elewaut,D.,Delsing,D.,van Loo, PF and Hendriks,RW(2012)。Btk水平设置了小鼠B细胞活化和自身反应性B细胞阴性选择的阈值。血液119(16):3744-3756。
Li ,BWS,Beerens,D.,Brem,MD和Hendriks,RW(2017)。小鼠过敏性气道炎症模型中第2组先天淋巴样细胞的特征。方法分子生物学1559:169-183。  
伊利诺伊州Martensson,N.Almqvist,O.Grimsholm和AI的Bernardi(2010)。B前细胞受体检查点。FEBS Lett 584(12):2572-2579。
Nishizumi,H.,Taniuchi,I.,Yamanashi,Y.,Kitamura,D.,Ilic,D.,Mori,S.,Watanabe,T。和Yamamoto,T。(1995)。lyn缺陷小鼠外周B细胞的增殖受损和自身免疫性疾病的迹象。免疫力3(5):549-560。
Pieper,K.,Grimbacher,B.和Eibel,H.(2013年)。B细胞生物学与发展。过敏临床免疫杂志131(4):959-971。
Rip,J.,de Bruijn,MJW,Kaptein,A.,Hendriks,RW和Corneth,OBJ(2020)。用于人和鼠B细胞亚群中信号研究的Phosphoflow方案。免疫学杂志204(10):2852-2863。
Rip,J.,Van Der Ploeg,EK,Hendriks,RW和Corneth,OBJ(2018)。牛逼布鲁顿酪氨酸他的作用在免疫细胞信号和系统性自身免疫激酶。免疫评论38(1):17-62。
Saito,K.,Scharenberg,AM和Kinet,JP(2001)。Btk PH域和磷脂酰肌醇-3,4,5-三磷酸之间的相互作用直接调节Btk。生物化学杂志276(19):16201-16206。
Tonegawa,S。(1983)。体细胞产生抗体多样性。自然302(5909):575-581。
Wang,D.,Feng,J.,Wen,R.,Marine,JC,Sangster,MY,Parganas,E.,Hoffmeyer,A.,Jackson,CW,Cleveland,JL,Murray,PJ and Ihle,JN(2000) )。磷脂酶Cgamma2在B细胞和几种Fc受体的功能中至关重要。免疫13(1):25-35。
登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用:Rip, J., Hendriks, R. W. and Corneth, O. B. (2021). A Versatile Protocol to Quantify BCR-mediated Phosphorylation in Human and Murine B Cell Subpopulations. Bio-protocol 11(3): e3902. DOI: 10.21769/BioProtoc.3902.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。