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Feb 2020

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Protocol for Isolation, Stimulation and Functional Profiling of Primary and iPSC-derived Human NK Cells
原代和iPSC来源的人类NK细胞的分离、刺激和功能分析方案   

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Abstract

Natural killer (NK) cells are innate immune cells, characterized by their cytotoxic capacity, and chemokine and cytokine secretion upon activation. Human NK cells are identified by CD56 expression. Circulating NK cells can be further subdivided into the CD56bright (~10%) and CD56dim NK cell subsets (~90%). NK cell-like cells can also be derived from human induced pluripotent stem cells (iPSC). To study the chemokine and cytokine secretion profile of the distinct heterogenous NK cell subsets, intracellular flow cytometry staining can be performed. However, this assay is challenging when the starting material is limited. Alternatively, NK cell subsets can be enriched, sorted, stimulated, and functionally profiled by measuring secreted effector molecules in the supernatant by Luminex. Here, we provide a rapid and straightforward protocol for the isolation and stimulation of primary NK cells or iPSC-derived NK cell-like cells, and subsequent detection of secreted cytokines and chemokines, which is also applicable for a low number of cells.

Keywords: CD56 (CD56), Natural killer cells (自然杀伤细胞), Induced pluripotent stem cells (诱导多能干细胞), Cytokines (细胞因子), Chemokines (化学因子), Peripheral blood (外周血), Luminex ( Luminex)

Background

Natural killer (NK) cells are part of the innate immune system and provide the first line defense against viral infections and malformations. In the human blood, two distinct NK cell populations can be identified based on CD56 and CD16 expression: CD56brightCD16+/- and CD56dimCD16+ NK cells (Melsen et al., 2016). CD56bright NK cells represent the minor subset (~10% of NK cells) and are known for their cytokine and chemokine secretion, but low cytotoxicity. In contrast, the CD56dim NK cells have high cytotoxic capacity. To test functional responses of NK cells in the absence of other cells, NK cells first need be enriched from peripheral blood mononuclear cells (PBMC) by negative enrichment. To study the production of effector molecules by the distinct NK cell populations upon stimulation, intra- and extracellular flow cytometry can be performed (Eberlein et al., 2010). However, this technique is limited to the number of effector molecule-specific antibodies available. Moreover, multiple samples are required to analyze the major effector molecules making this technique less suitable for a low number of cells. As an alternative, the distinct NK cell subsets can be sorted and stimulated. Cytokines and chemokines can be subsequently measured in the supernatant by Luminex (Lugthart et al., 2016). The advantages are: 1) distinct NK cell subsets cannot influence each other, 2) the supernatant can be harvested at multiple timepoints, which allows studying kinetics of the same cells, 3) > 25 effector molecules can be studied at once. Moreover, since no cell harvesting and fixation is required, cells could be stored or used for further experiments.


As an alternative for primary NK cells, NK cells can be derived from human iPSC. The different protocols used are based on the stepwise differentiation of human iPSC into mesoderm, hemogenic or hematopoietic progenitor cells, and subsequently into CD56+ NK cells. CD34+CD45+ hematopoietic progenitors or CD34+CD31+ hemogenic progenitors are generated using either stromal cells like OP9 or embryoid bodies in the presence of hematopoietic and vascular growth factors (Knorr et al., 2013). The CD34+ progenitors are further differentiated towards NK cells using a cytokine cocktail in either the presence or absence of OP9-DL1 stroma cells (Knorr et al., 2013; Zeng et al., 2017). Cytokine cocktails used contain SCF, FLT3L, IL-3, IL-15 and IL-7 in the absence of OP9-DL1 (Knorr et al., 2013), or SCF, FLT3L, and IL-7 when co-culturing on OP9-DL1 cells (Zeng et al., 2017). The latter conditions simultaneously generate T cells (Themeli et al., 2013 and 2020), however, substitution of IL-7 by IL-15, or the addition of IL-15 results in much purer (> 99%) CD56+ NK cell populations (Zeng et al., 2017). In our hands, CD56+ NK cells generated from iPSC best resemble primary CD56bright NK cells (Themeli et al., 2020). If the generation of T cells is hampered, by example in the case of RAG2 deficiency, previously undescribed small populations with NK cell-specific cytokine secretion profiles can be found (Themeli et al., 2020). To functionally profile rare NK cell-like populations a sensitive and easy-to-use protocol is paramount. Here we describe such a protocol that allows a rapid assessment of NK cell-specific secretion profiles using as few as 10,000 cells.


Materials and Reagents

NK cell isolation
General

  1. Laboratory disposables:

    Pipettes

    15 ml tubes

    50 ml tubes (Greiner, Cellstar, catalog number: 227261)

    Pasteur pipettes

    Eppendorf tubes (Eppendorf, catalog number: 0030121023)

  2. 50 ml Syringe (Becton Dickinson Medical, catalog number: BD300865), 10 ml Syringe (Becton Dickinson Medical, catalog number: BD 307736)

  3. 0.22 µm syringe filter (Whatman FP30 CA-S, catalog number: 10462300)

  4. Syringe needle (Becton Dickinson MICROLANCE 3, 19 G x 40 MM, catalog number: 301500)

  5. 96-wells round bottom plate non-sterile (Corning, catalog number: 3799)

  6. Micronic tubes (NBS scientific, catalog number: MP32022)

  7. 5 ml round-bottom polystyrene tubes (Corning, Falcon, catalog number: 352052)

  8. PBS (Fresenius Kabi, catalog number: 8717973380153), 4°C

  9. Bürker-Türk counting chamber (VWR, catalog number: HECH40444702)

  10. Türk′s solution (Merck, Sigma-Aldrich, catalog number: 1092770100 )

  11. Bovine serum albumin (BSA) (Merck,Sigma-Aldrich, catalog number: A9576), 4 °C

  12. EDTA (Merck, Calbiochem, catalog number: 324503, Molecular weight 372.24), RT

  13. Distilled water (Aqua BBraun, B Braun, catalog number: 0082479E)

  14. NaOH (Merck, catalog number: 106498)

  15. Fetal Calf Serum (FCS, heat inactivated for 30 min at 56 °C to inactivate complement) (Merck, Sigma-Aldrich, catalog number: F7524), -20°C

  16. EDTA solution 0.5 M (see Recipes), RT

  17. AIMV medium (Thermo Fisher Scientific, Gibco, catalog number: 31035025), 4 °C

  18. Collection medium (see Recipes), 4 °C

  19. Antibody for cell sorting (see Recipes), 4 °C

    CD56 clone N901 (ECD) (Beckman Coulter, for instance, catalog number: A82943), 4 °C or clone 5.1H11 (Biolegend, for instance, catalog number: 362550), 4°C or clone B159 (Becton Dickinson, for instance, catalog number: 560361), 4 °C


Only required for primary NK cell isolation

  1. Pre-separation filters 30 µm (Miltenyi, catalog number: 130-041-407), RT

  2. MACS columns MS or LS (Miltenyi, catalog number: 130-042-201 or 130-042-401), RT

  3. Ficoll Paque Plus (Merck, Sigma, catalog number: GE17-1440-02), RT, dark

  4. RPMI 1640 Medium (Thermo Fisher Scientific, Gibco, catalog number: 72400054), 4 °C

  5. Human serum albumin 200 g/L (Sanquin, Albuman, catalog number: 8717185830897) 4 °C

  6. Penicillin-Streptomycin 100x (Merck, Sigma-Aldrich, catalog number: P0781), -20 °C

  7. NK cell isolation kit human (Miltenyi, catalog number: 130-092-657), 4 °C

  8. CD33 clone P67.6 (PE) (Becton Dickinson, for instance, catalog number: 345799), 4 °C

  9. CD14 clone M5E2 (PE-Cy7) (Becton Dickinson for instance, catalog number: 557742), 4 °C

  10. CD3 clone UCHT1 (BV421) (Becton Dickinson, for instance, catalog number: 562426), 4 °C

  11. CD19 clone SJ25C1 (BV510) (Becton Dickinson, for instance, catalog number: 562947), 4 °C

  12. MACS buffer (see Recipes), 4 °C

  13. Dilution medium (see Recipes), 4 °C

  14. Wash medium (see Recipes), 4 °C


Only required for iPSC-derived NK cell isolation

  1. 30 µm CellTrics filter (Sysmex, catalog number: 04-004-2326)

  2. CD7 clone 124-1D1 (eBioscience, for instance, catalog number: 25-0079-41), 4 °C or clone 4H9 (Becton Dickinson, for instance, catalog number: 347483), 4 °C or clone M-T701 (Becton Dickinson, catalog number: 561934), 4 °C


    Stimulation

  1. Laboratory disposables:

    Pipettes

    15 ml tubes

    50 ml tubes

    Eppendorf tubes

  2. 96-well round bottom plate (Greiner, catalog number: 650185)

  3. AIMV medium (Thermo Fisher Scientific, Gibco, catalog number: 31035025)

  4. Fetal Calf Serum (FCS, heat inactivated for 30 min at 56 °C to inactivate complement) (Merck, Sigma-Aldrich, catalog number: F7524), -20 °C

  5. Recombinant human IL-12 (Peprotech, catalog number: 200-12), -20 °C

  6. Recombinant human IL-15 (Peprotech, catalog number: 200-15), -20 °C

  7. Recombinant human IL-18 (MBL International, catalog number: B001-5), -20 °C

  8. Interleukin mix (see Recipes)


    Functional profiling

  1. Laboratory disposables:

    Pipettes

    15 ml polypropylene tubes

    0.5 ml polypropylene tube

    Reagent reservoirs

  2. Aluminium foil

  3. Sealing tape (for instance, Merck, Greiner, catalog number: A5596-100EA)

  4. Ice

  5. Paper towels

  6. Bio-Plex Pro Human Cytokine 27-plex Immunoassay (Bio-Rad, catalog number: M50-0KCAF0Y), 4 °C

Equipment

  1. Laminar flow cabinet for sterile work (biosafety level II) (Euroflow EF4, CleanAir by Baker)

  2. Tube rack to hold 15, 50 ml tubes (for instance, VWR, catalog numbers: 89215-778 and 89215-778)

  3. Micropipettes (P10, P100, P1000) (for instance, Gilson, catalog number: F167380)

  4. 12 channel multichannel pipette (for instance, Eppendorf, catalog number: 3125000060)

  5. Table top centrifuge with adapters for plates and for 15 ml and 1.5 ml tubes (Eppendorf, model: 5810 R)

  6. CO2 Incubator (at 5% CO2 and 37 °C) (for instance, Panasonic, model: MCO 170-AIC)

  7. Magnetic stirrer (for instance, VWR, catalog number: 89215-778)

  8. FACS Aria cell sorter (Becton Dickinson, Aria I, II, III) but any equivalent fluorescence-activated cell sorter should work

  9. MiniMACS or MidiMACS separator (Miltenyi, catalog number: 130-042-102 or 130-042-302, respectively)

  10. MACS MultiStand (Miltenyi, catalog number: 130-042-303, RT)

  11. Autoclave (for instance, VWR, Ward’s, catalog number: 470230-598)

  12. Vortex (for instance Scientific Industries, model: Vortex Genie 2, catalog number: 200-SI-0236)

  13. Bio-Plex 200 system (Bio-Rad, for instance, catalog number: 171000201)

  14. Bio-Plex handheld magnetic washer (Bio-Rad, catalog number: 171020100)

  15. Plate shaker (for instance, Biosan, model: PSU-2T)

Software

  1. Diva software (Becton Dickinson, v6.0 or later, 2007 or later)

  2. Bio-Plex Manager software (Bio-Rad, v6.2, 2018)

Procedure

Primary NK cell isolation

  1. Peripheral blood mononuclear cell (PBMC) isolation

    Note: At room temperature (RT) unless stated otherwise.

    1. Dilute at least 15 ml blood 2x in dilution medium and mix.

      Note: From 15 ml blood typically ~1.5 x106 NK cells can be isolated. This number will yield ~7.5 x 104 CD56bright NK cells.

    2. Add 4 ml Ficoll to a 15 ml tube.

    3. Carefully layer the diluted blood sample (10 ml) onto the Ficoll (do not mix).

    4. Repeat Steps 2 and 3 for the remaining diluted blood.

    5. Centrifuge 15 min at 1,000 x g without brake.

    6. Harvest the PBMC by carefully pipetting (using a Pasteur pipette) the white layer of cells between plasma and ficoll.

    7. Transfer the PBMC to a new 50 ml tube (pool per sample max 25 ml).

    8. Add wash medium up to 50 ml.

    9. Centrifuge 10 min at 800 x g.

    10. Remove the supernatant by aspiration and resuspend the cells.

    11. If applicable, pool the cells in 150 ml tube.

    12. Add wash medium up to 10 ml.

    13. Centrifuge 10 min at 540 x g.

    14. Remove supernatant and resuspend cells in the recommended volume of MACS buffer (~1 ml MACS buffer for every 10 ml of undiluted blood).

    15. Count the cells with Türk′s solution (dilute 10 µl cell suspension with 90 µl Türk′s solution). Keep the remaining cells at 4 °C.

    16. Keep 2 x 106 PBMC aside for single stain controls in sorting procedure.


  2. NK cell isolation from PBMC using MACS

    Note: Keep cells and buffers at 4 °C during the NK cell isolation. Check the manufacturer’s instructions for any updates in the protocol.

    1. Transfer cells to 15 ml tube and add up to 14 ml MACS buffer.

    2. Centrifuge 5 min 540 x g.

      Note: Volumes for magnetic labeling are minimum volumes, when working with less than 1 x 107 cells, use this volume, when working with more than 1 x 107 cells, scale up the volumes accordingly.

    3. Remove supernatant by aspiration. Resuspend cells in 40 µl of MACS buffer per 1 x 107 cells.

      Example: For 1.5 x 107 cells, add 60 µl of MACS buffer

    4. Add 10 µl of NK cell Biotin-Antibody Cocktail per 1 x 107 cells.

    5. Mix well and incubate for 5 min at 4 °C.

    6. Add 30 µl of MACS buffer per 1 x 107 cells.

    7. Add 20 µl of NK cell MicroBead Cocktail per 1 x 107 cells.

    8. Mix well and incubate for 10 min at 4 °C.

    9. Place the column (MS for ≤ 1 x 107 cells, LS for > 1 x 107 PBMC) in the magnetic field of the MACS separator, which is placed on the MACS MultiStand. A maximum of 1 x 108 PBMC can be loaded per LS column.

    10. Put the pre-separation filter on top of the column.

    11. Position the column into a 15 ml tube.

    12. Rinse the column by pipetting MACS buffer in the filter: 500 µl (MS column), 3 ml (LS column)

    13. Replace the 15 ml tube filled with buffer with a new 15 ml tube (the flow-through with NK cells will be collected in this tube).

      Important: Only pipet new volumes when the column reservoir is empty.

    14. Pipette the labeled PBMC in the filter. A minimum volume of 500 µl is required for magnetic separation. If necessary, add MACS buffer to the cell suspension.

    15. Wash column by pipetting MACS buffer in the filter: 500 µl (MS column), 3 ml (LS column)

    16. Repeat Step 15 twice.

    17. Centrifuge the 15 ml tube containing the unlabeled NK cells and centrifuge 5 min at 540 x g.

    18. Resuspend in MACS buffer (~500-1,000 µl) and count.


  3. FACS of NK cell subsets

    Note: Keep cells and buffers at 4 °C.

    1. Prepare antibody mixes (30 µl per 1 x 106 NK cells). Prevent bleaching by keeping the antibody mixes in the dark! See Recipes for example calculation. Important: use titrated antibodies.

      Note: Titration is performed on 106 PBMC using the recommended dilution as the median of 5 concentrations. A 2-fold dilution factor is typically applied. Recommended dilutions should be saturating but at the same not alter the mean fluorescence intensity of the antigen negative cells.

    2. Prepare single stain control mixes (1 antibody + MACS buffer), 25 µl per single stain. Use same dilution as used for complete antibody mix. Do not forget to include 1 unstained control.

    3. Transfer 2 x 105 PBMC into a well of a 96 wells plate (1 well/single stain).

    4. Pellet cells by centrifugation for 5 min, 540 x g at 4 °C (for tube), or 2 min, 540 x g at 4 °C (for plate).

    5. For tube: remove supernatant from the 15 ml tube by aspiration and resuspend in the correct amount of antibody mix. Incubate in the dark for 30 min at 4 °C.

      For plate: remove supernatant from the wells by flicking the plate once above the sink. Remove any access liquid by gently tapping the plate once onto a paper towel. Add 25 µl of single stain mix. Incubate in the dark for 30 min at 4 °C.

    6. Wash cells by adding 10 ml (for tube) and 200 µl (for plate) MACS buffer to the cells.

    7. Pellet cells by centrifugation as before: 5 min, 540 x g at 4 °C (for tube), 2 min, 540 x g at 4 °C (for plate).

    8. Remove supernatant as described before (Step 5).

    9. For tube: resuspend NK cells in MACS buffer, to a concentration of 1 x 107 NK cells per ml.

      For plate: resuspend the PBMC in 70 µl MACS buffer, and collect them in micronic tubes.

    10. Prepare round bottom polystyrene tubes with 3 ml collection medium (max. 6 x 105 cells can be collected in 1 tube).

    11. Sort the cells at low pressure (100 µm nozzle, 20 PSI, this is important for the functioning of the NK cells after the sorting). Use the gating strategy as depicted in Figure 1. If more subsets are desired, the CD56bright can be further subdivided in a CD16- and CD16+ subset. Sorting of 1.5 x 106 NK cells will yield (when taking into account cell loss) ~7.5 x 104 CD56bright NK cells and 1 x 106 CD56dim NK cells.



      Figure 1. Gating strategy for sorting of primary NK cell subsets. First, lymphocytes are gated based on forward- and side scatter. Next, doublets, CD3+ T cells, CD19+ B cells, CD14+ monocytes, and CD33+CD56- myeloid cells are excluded. NK cells can be further subdivided based on intensity expression of CD56 and CD16: CD56brightCD16+/- and CD56dimCD16+ NK cells.


    iPSC-derived NK cell isolation by FACS
    Note: Keep cells and buffers at 4 °C during the NK cell isolation.
  1. Collect the floating cells in the iPSC differentiation culture (Figure 2A) into a 15 ml or 50 ml conically shaped tube. The tube size depends on the scale of differentiation (typical format: a few wells from a 6-well plate).

    Note: ~1 x 106 floating cells are typically harvested from one 6 well plate. Depending on the success of differentiation 20-70% of the floating cells are CD7+CD56+ NK cells.

  2. Pass the cell suspension through a 30 µm CellTrics filter (Sysmex) and collect the cells in a 15 ml or 50 ml conically shaped tube.

  3. Centrifuge the collected cell suspension at 400 x g for 5 min at 4 °C.

  4. Remove the supernatant by aspiration and resuspend the pelleted cells in 500 µl ice-cold PBS (wash Step 1).

  5. Centrifuge the cells at 400 x g for 5 min at 4 °C.

  6. Remove the supernatant by aspiration and resuspend the pelleted cells in 500 µl ice-cold MACS buffer (wash Step 2).

  7. Centrifuge the cells at 400 x g for 5 min at 4 °C.

  8. Remove the supernatant by aspiration and resuspend the pelleted cells in 500 µl ice-cold MACS buffer (wash Step 3).

  9. Take a small aliquot (10 µl) and count the cells.

  10. Transfer the cells into a 0.5 ml Eppendorf tube or 96 wells pate (round-bottom or V-shaped bottom well). Required number of tubes/wells: unstained control, single stain controls, complete stain. Use 2 x 104-5 x 104 cells for the single stain or unstained controls. The rest is used for the combi-stain of the cells that will be sorted. Never go beyond 1 x 106 cells/ 25 µl staining solution.

  11. Centrifuge the cells at 400 x g for 5 min at 4 °C (for tube) or 2 min, 540 x g at 4 °C (for plate).

  12. Prepare antibody mixes: single stain control mixes (1 antibody + MACS buffer), 25 µl per single stain, and a complete antibody mix containing at least anti-CD7 and anti-CD56 to identify NK cell-like cells.

    Note: Use identical dilutions for the same antibodies and shield the antibodies from light, particularly, when tandem dyes are used as conjugates. Do not forget to include an unstained control and preferentially also a positive control consisting of mononuclear blood cells, by example from peripheral blood.

  13. Remove the supernatant by aspiration ( fortube) or by flicking the plate once above the sink (for plate). Remove any access liquid by gently tapping the plate once onto a paper towel (for plate).

  14. Resuspend the cells in the antibody mix.

  15. Incubate in the dark for 30 min at 4 °C (typically in the fridge).

  16. Centrifuge the cells at 400 x g for 5 min at 4 °C (for tube) or 2 min, 540 x g at 4 °C (for plate).

  17. Remove the supernatant as described before (Step 13) and resuspend the cells in 200 µl ice-cold MACS buffer.

  18. Centrifuge the cells at 400 x g for 5 min at 4 °C (for tube) or 2 min, 540 x g at 4 °C (for plate).

  19. Remove the supernatant as described before (Step 13) and resuspend the single stain, unstained cells, and positive control cells in 70 µl MACS buffer and transfer them into Micronic or 5 ml round-bottom polystyrene tubes. The cells to be sorted are resuspended at a concentration of 10 x 106 cells per ml.

  20. Prepare 5 ml round-bottom polystyrene tubes with 3 ml collection medium (max 6 x105 cells can be collected in 1 tube).

  21. Sort the CD7+CD56+ cells at low pressure (100 µm nozzle, 20 PSI). Use the gating strategy as depicted in Figure 2B.

    Note: Dependent on the question to be addressed and required purity antibodies against additional NK cell markers such as CD16 may be used.



    Figure 2. Isolation of iPSC-derived NK cells by FACS. A. An example of the differentiation of iPSC towards NK cells after co-culturing with OP9-DL1 cells for 3 weeks (100x magnification). Non-adherent cells are harvested and CD56+ NK cells are isolated using FACS. B. Gating strategy to isolate iPSC-derived CD7+CD56+ NK cells.


    NK cell stimulation

  1. Spin down the sorted NK cell subsets and resuspend at a concentration of 5.6 x 104 cells/ml in AIMV medium with 1% Penicillin-Streptomycin (the final FCS concentration will be ~5%).

  2. Add 180 µl /well NK cells in a sterile 96-wells round-bottom plate.

  3. Add 20 µl of IL-12, IL-15 and IL-18 mix and medium without cytokines (AIMV + 1% Penicillin-Streptomcyin) to the different wells.

    Note: Cells from healthy donors that are cultured in IL-12, IL-15 and IL-18 are the positive control for IFN-γ production. Cells cultured in medium without cytokines act as negative control. Preferentially, stimulations should be performed in triplicate. If more cells were sorted than needed stimulations with single or double cytokine mixtures should be included.

  4. Include 1 well with 200 µl medium (AIMV + 1% Penicillin-Streptomcyin + 5% FCS) and 1 well with 200 µl medium supplemented with interleukins (as background for the Luminex).

  5. Wrap plastic foil around plate to prevent evaporation.

  6. Culture cells for 20 h in incubator at 37 °C, 5% CO2.

    Note: When studying kinetics, take a sample of 10 µl at multiple time points.

  7. Spin down plate, 540 x g for 2 min.

  8. Harvest supernatant and store in sealed 96 wells plate or Micronic tubes at -20 °C.


Luminex
Note: The protocol below has been optimized and therefore differs from the manufacture’s instruction.

  1. Spin down the standard vial and reconstitute a single vial of standard in 500 µl of diluent (= AIMV medium + 5% FCS), gently vortex for 5 s and incubate on ice for 30 min.

  2. Label 9 0.5 ml polypropylene tubes Std1-Std8 and Blank.

  3. Add the specified volume of diluent to each tube (Table 1).

  4. Vortex the reconstituted standard gently for 5 sec, take 128 µl and add to tube Std1.

  5. Vortex Std1 and transfer 50 µl from tube Std1 to tube Std2 and vortex. Important: use new pipet tips for every volume transfer.

  6. Continue with serial dilutions form tube S2 to S8 by transferring 50 µl each time.


    Table 1. Serial dilutions to generate a standard curve


  7. Thaw supernatant, and centrifuge at 1,000 x g for 4 min at RT.

  8. Transfer supernatant to clean polypropylene tube.

  9. Dilute samples 1:6 in diluent (once thawed keep samples on ice) and equilibrate to RT before use.

  10. Make a plate lay-out (for example Figure 3) to check the number of wells required. Use duplicates for the standards and blanks. Fill the plate vertically.



    Figure 3. Plate lay-out. An example of a plate lay-out with 8 standards and 1 blank in duplicate, and 6 samples.


  11. Add the required volume of assay buffer to a 15 ml tube (Table 2).

  12. Vortex the stock beads for 30 s at medium speed. Carefully open the cap and pipet any liquid trapped in the cap back into the vial. Important: do not centrifuge the stock beads, since the beads will be spun down.

  13. Dilute the beads by pipetting the required volume of stock beads into the 15 ml tube (Table 2). Important: protect the beads from light with aluminium foil.


    Table 2. Bead dilutions


  14. Bring standards and samples to RT before use.

  15. Vortex the diluted beads at medium speed for 30 s and pour the beads into a reagent reservoir. Transfer 50 µg of beads to each well of the flat bottom plate.

  16. Add 100 µl wash buffer per well. Position the plate for at least 60 s on the handheld magnet and quickly decant the waste solution. Remove any access liquid by tapping the plate onto a paper towel. Remove plate from the magnet, carefully resuspend the beads by tapping the plate, and repeat this washing step.

  17. Gently vortex the diluted standards, blanks and samples for 5 s. Transfer 50 µl to each well. Important: use a new pipette tip for each volume transfer.

  18. Cover the plate with a new sheet of sealing tape and protect from light with aluminum foil. Incubate on shaker at 450 rpm for 45 min at RT.

  19. Prepare the dilution of detection antibodies 10 min before use. Add the required volume of detection antibody diluent to a new 15 ml tube (Table 3).

  20. Vortex the stock detection antibodies at medium speed for 15-20 s. Spin down the stock for 30 s.

  21. Dilute the 10x detection antibodies by pipetting the required volume into the 15 ml tube (Table 3).

  22. After the 45 min incubation, remove the sealing tape and wash 3x with 100 µl washing buffer.

  23. Vortex the diluted detection antibodies for 5 s, pour into a reagent reservoir and transfer 12.5 µl to each well.

  24. Cover plate with a new sealing tape and protect from light with aluminum foil. Incubate on shaker at 450 rpm for 30 min at RT.


    Table 3. Dilution of detection antibodies

  25. Prepare the streptavidin-PE dilution 10 min before use. Add the required volume of assay buffer to a new 15 ml tube (Table 4).


    Table 4. Dilution streptavidin-PE

  26. Vortex the 100x SA-PE for 5 s at medium speed and spin down for 30 s.

  27. Pipette the required volume of SA-PE to the 15 ml tube (Table 4). Vortex and protect from light.

  28. Power-up the Bio-plex system (30 min in advance).

  29. After the 30 min incubation of the detection antibody, remove the sealing tape and wash the plate 3x with 100 µl washing buffer.

  30. Vortex the diluted SA-PE for 5 s, pour into a reagent reservoir, and transfer 25 µl to each well.

  31. Cover the plate with sealing tape and aluminum foil. Incubate on shaker at 450 rpm for 10 min at RT.

  32. Wash the plate 3x with 100 µl wash buffer.

  33. To resuspend beads for plate reading, add 80 µl of assay buffer to each well and cover the plate with sealing tape. Incubate on shaker at 850 ± 50 rpm for 30 s at RT.

  34. Remove the tape and read the plate (using low PMT settings). In case you expect low concentrations, use high PMT settings.

  35. Data analysis: For a detailed handbook we refer to online tutorials by Bio-Rad (Reference 1 for instance).

    Note: It is important to subtract the background values (the wells with only medium or medium supplemented with interleukins) from the other values, which are automatically generated by the software based on the standard curve. In addition, check whether the experiment was successful by verifying that the negative control (cells without stimulation) is truly negative, and that the positive control (cells stimulated with IL-12, IL-15 and IL-18) shows IFN-γ production. An example of cytokine production by a variety of NK cells is shown in Figure 4.



    Figure 4. Example of Luminex measurements of stimulated primary and iPSC-derived NK cells. The Bio-Plex Pro Human Cytokine 27-plex Immunoassay allows detection of 27 cytokines/chemokines. NK cells are producers of predominantly GM-CSF, TNF-α and IFN-γ and various chemokines. GM-CSF, TNF-α and IFN-γ production is shown upon the culture of primary NK cell subsets (CD56bright and CD56dim) and iPSC-derived NK cells (CD7+CD56+) in the presence of IL-12, IL-15 and IL-18 for 20 h. The data is a representative of at least 2 independent experiments. The means and standard deviations are shown.

Recipes

  1. Dilution medium

    500 ml RPMI 1640 medium

    5 ml penicillin-streptomycin 100x

    Add Penicillin-Streptomycin to RPMI 1640 medium and shake

  2. Wash medium

    500 ml RPMI 1640 medium

    5 ml Penicillin-Streptomycin 100x

    2 ml Human serum albumin 200 g/L (final = 0.8 g/L)

    Add Penicillin-Streptomycin and human serum albumin to RPMI 1640 medium and shake

  3. EDTA solution 0.5 M

    1,000 ml distilled water

    186.12 g EDTA

    Add EDTA to a bottle with distilled water, add magnetic stirring bar, and put the bottle on a magnetic stirrer. Add pellets NaOH until EDTA dissolves (around pH 7.5-8). Autoclave and store de bottle at RT. Use a syringe filter 0.22 µm to filter the solution before use

  4. MACS buffer

    500 ml PBS

    8.3 ml 30% BSA (final = 0.5%)

    2 ml 0.5 M EDTA solution (final = 2 mM)

    Add BSA and filtered EDTA solution to the PBS and mix

  5. Antibody mix for primary NK cells

    MACS buffer

    1:20 CD33 PE

    1:200 CD16 FITC

    1:100 CD56 ECD

    1:100 CD14 PE-Cy7

    1:200 CD3 BV421

    1:100 CD19 BV510

    Add the antibodies to the MACS buffer (in an Eppendorf tube) and vortex. Keep the mix in the dark at 4 °C

  6. Collection medium

    AIMV medium

    30% FCS

    1% penicillin-streptomycin

    Add the FCS and Penicillin-Streptomycin to the AIMV medium and mix

  7. Interleukin mix

    AIMV medium

    1% Penicillin-Streptomycin

    10 ng/ml IL-12

    10 ng/ml IL-15

    20 ng/ml IL-18

    Add the Penicillin-Streptomycin and interleukins to the AIMV medium and vortex

Acknowledgments

This study was supported by the graduate program of NWO (J.M.), Leiden University Medical Center fellowship (J.M.), LSBR foundation (H.M.) (LSBR09-11), the European Commission (Marie Curie Individual Fellowship to M.T.), the Dutch Cancer Society (KWF) (M.T.), and Stichting VUmc CCA (M.T.).

Competing interests

All authors have declared to have no competing interests.

Ethics

Primary NK cells were isolated from buffy coats of healthy adult donors (Sanquin Blood bank, Region Southwest, Rotterdam, The Netherlands). Human materials for iPSC were collected according to the approval by the “Medical Ethics Committees” of the Erasmus MC (MEC-2016-606) or the LUMC (P13-080). The experiments involving human materials were done in accordance with the principles outlined in the “Declaration of Helsinki”.

References

  1. Bio-Plex ManagerTM and Data ProTM Software: Streamline Multiplex Data Analysis (2016). YouTube, 1 Feb.https://www.youtube.com/watch?v=v2i3Vp6MwyE.
  2. Eberlein, J., Nguyen, T. T., Victorino, F., Golden-Mason, L., Rosen, H. R. and Homann, D. (2010). Comprehensive assessment of chemokine expression profiles by flow cytometry. J Clin Invest 120(3): 907-923.
  3. Knorr, D. A., Ni, Z., Hermanson, D., Hexum, M. K., Bendzick, L., Cooper, L. J., Lee, D. A. and Kaufman, D. S. (2013). Clinical-scale derivation of natural killer cells from human pluripotent stem cells for cancer therapy. Stem Cells Transl Med 2(4): 274-283.
  4. Lugthart, G., Melsen, J. E., Vervat, C., van Ostaijen-Ten Dam, M. M., Corver, W. E., Roelen, D. L., van Bergen, J., van Tol, M. J., Lankester, A. C. and Schilham, M. W. (2016). Human Lymphoid Tissues Harbor a Distinct CD69+CXCR6+ NK Cell Population. J Immunol 197(1): 78-84.
  5. Melsen, J. E., Lugthart, G., Lankester, A. C. and Schilham, M. W. (2016). Human Circulating and Tissue-Resident CD56bright Natural Killer Cell Populations. Front Immunol 7: 262.
  6. Themeli, M., Chhatta, A., Boersma, H., Prins, H. J., Cordes, M., de Wilt, E., Farahani, A. S., Vandekerckhove, B., van der Burg, M., Hoeben, R. C., Staal, F. J. T. and Mikkers, H. M. M. (2020). iPSC-Based Modeling of RAG2 Severe Combined Immunodeficiency Reveals Multiple T Cell Developmental Arrests. Stem Cell Reports 14(2): 300-311.
  7. Themeli, M., Kloss, C. C., Ciriello, G., Fedorov, V. D., Perna, F., Gonen, M. and Sadelain, M. (2013). Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy. Nat Biotechnol 31(10): 928-933.
  8. Zeng, J., Tang, S. Y., Toh, L. L. and Wang, S. (2017). Generation of“Off-the-Shelf” Natural Killer Cells from Peripheral Blood Cell-Derived Induced Pluripotent Stem Cells. Stem Cell Reports 9: 1796-1812.

简介

[摘要]天然杀伤(NK)细胞是先天性免疫细胞,其特征在于其细胞毒性能力以及活化后的趋化因子和细胞因子分泌。人NK细胞通过CD56表达鉴定。循环的NK细胞可进一步细分为CD56亮(约10%)和CD56暗NK细胞亚群(约90%)。NK细胞样细胞也可以源自人诱导的多能干细胞(iPSC)。为了研究不同的异源NK细胞亚群的趋化因子和细胞因子分泌概况,可以进行细胞内流式细胞仪染色。然而,当起始原料有限时,该测定法具有挑战性。或者,可以通过Luminex测量上清液中分泌的效应子分子来富集,分选,刺激和功能性分析NK细胞亚群。在这里,我们提供了一种快速直接的方案,用于分离和刺激原代NK细胞或iPSC衍生的NK细胞样细胞,并随后检测分泌的细胞因子和趋化因子,这也适用于少量细胞。


[背景]自然杀伤(NK)细胞是先天免疫系统的一部分,提供第一线防御病毒感染和畸形。在人血中,可以基于CD56和CD16表达鉴定出两个不同的NK细胞群体:CD56明亮的CD16 +/-和CD56暗的CD16 + NK细胞(Melsen等,2016)。CD56亮的NK细胞代表次要的子集(约占NK细胞的10%),以其细胞因子和趋化因子的分泌而著称,但细胞毒性较低。相反,CD56昏暗的NK细胞具有高细胞毒性能力。为了在不存在其他细胞的情况下测试NK细胞的功能反应,首先需要通过负富集从外周血单核细胞(PBMC)富集NK细胞。为了研究刺激后不同的NK细胞群体产生效应分子的方法,可以进行细胞内和细胞外流式细胞术(Eberlein等,2010)。但是,该技术限于可获得的效应分子特异性抗体的数量。而且,需要多个样品来分析主要的效应分子,从而使该技术不太适合少量细胞。作为替代方案,可以分类和刺激不同的NK细胞亚群。随后可以通过Luminex在上清液中测量细胞因子和趋化因子(Lugthart等,2016)。的优点是:1)明显的NK细胞亚群不能相互影响,2)将上清液可以在多个时间点,这允许研究的相同细胞,3)的动力学收获> 25个效应分子可以一次进行研究。而且,由于不需要收集和固定细胞,因此可以储存细胞或将其用于进一步的实验。

作为原代NK细胞的替代物,NK细胞可以源自人iPSC。所使用的不同方案基于人iPSC逐步分化为中胚层,造血或造血祖细胞,然后分化为CD56 + NK细胞。CD34 + CD45 +造血祖细胞或CD34 + CD31 + hemogenic祖细胞使用的是基质细胞等OP9或在造血和血管生长因子的存在胚状体产生(克诺尔等人,2013年)。在存在或不存在OP9-DL1基质细胞的情况下,使用细胞因子混合物将CD34 +祖细胞进一步分化为NK细胞(Knorr等人,2013; Zeng等人,2017)。在没有OP9-DL1的情况下使用的细胞因子混合物包含SCF,FLT3L,IL-3,IL-15和IL-7 (Knorr等人,2013),或在OP9上共培养时包含SCF,FLT3L和IL-7 -DL1细胞(Zeng等,2017)。后一种条件同时产生T细胞(Themeli等人,20 13和20 20 ),但是,用IL-15替代IL-7或添加IL-15会导致CD56纯度更高(> 99%)。+ NK细胞群体(Zeng等,2017)。在我们手中,CD56 +从iPSC集生成的NK细胞BES吨类似于初级CD56明亮的NK细胞(Themeli等人2020 )。如果例如在RAG2缺乏的情况下阻碍了T细胞的产生,则可以发现先前未描述的具有NK细胞特异性细胞因子分泌特征的小群体(Themeli等,2020)。到泛函升升ý轮廓稀有NK细胞样群小号灵敏且易于使用的协议是极为重要的。在这里,我们描述了这样一种协议,该协议允许使用少至10,000个细胞快速评估NK细胞特异性分泌谱.

关键字:CD56, 自然杀伤细胞, 诱导多能干细胞, 细胞因子, 化学因子, 外周血, Luminex

材料和试剂

NK细胞分离

ģ ENERAL

1.实验室disposabl ES :        

P ipettes

15毫升管

50 ml管(Greiner,Cellsta r,货号:227261)

巴斯德移液器

E ppendorf管(Eppendorf,目录号:0030121023 )

2. 50毫升注射器(Becton Dickinson Medical,目录号:BD300865),10毫升注射器(Becton Dickinson Medical,目录号:BD 307736)        

3. 0.22 µm针筒式过滤器(Whatman FP30 CA-S,目录号:10462300)        

4.注射器针头(Becton Dickinson MICROLANCE 3,19 G x 40 MM,目录号:301500)        

5. 96孔圆形无菌底板(Corning,目录号:3799)        

6.微型电子管(NBS科学版,目录号:MP3 2022)        

7. 5毫升圆底聚苯乙烯管(Corning,Falcon,目录号:352052)        

8. PBS (Fresenius Kab i,目录号:8717973380153 ),4°C        

9. BURKER-谢蒂尔克计数室(VWR,目录号:HECH40444702)        

10.土耳其人的溶液(Merck公司,Sigma-Aldrich公司,目录号:1092770100)    

11.牛血清白蛋白(BSA)(Merck,Sigma - Aldrich,目录号:A9576 ),4 °C    

12. EDTA(默克,Calbiochem公司,产品目录号:324503,分子量372.24 ),RT    

13.蒸馏水(Aqua BBraun,B Braun,目录号:0082479E)    

14. NaOH(Merck,目录号:106498)    

15.胎牛血清(FCS,在56 °C加热30分钟以灭活补体)(Merck,Sigma - Aldrich,目录号:F7524 ),- 20°C    

16. EDTA溶液0.5 M(请参见“配方” ),RT    

17. AIMV介质(Thermo Fisher Scientific,Gibco,目录号:31035025 ),4 °C    

18.收集介质(请参见食谱),4 °C    

19.细胞分选抗体(参见食谱),4 °C    

CD56克隆N901(ECD)(例如Beckman Coulter,目录号:A82943 ),4 °C或克隆5.1H11(例如Biolegend ,目录号:362550 ),4°C或克隆B159(例如Becton Dickinson),目录号:560361 ),4°C

 

仅对于原代NK细胞分离是必需的

预分离过滤器30 µm(Milteny i,目录号:130-041-407 ),RT

MACS列MS或LS(Miltenyi,目录号:130-042-201或130-042-401 ),RT

Ficoll Paque Plus(Merck,Sigma,目录号:GE17-1440-02 ),RT,黑色

RPMI 1640介质(Thermo Fisher Scientific,Gibco,目录号:72400054 ),4 °C

人血清白蛋白200克/升(桑昆,白蛋白,目录号:8717185830897 )4 °C

青霉素-链霉素100x(Merck,Sigma - Aldrich,目录号:P0781 ),- 20°C

人类NK细胞分离试剂盒(Miltenyi,目录号:130-092-657 ),4 °C

CD33克隆P67.6(PE)(例如Becton Dickinson ,目录号:345799 ),4 °C

CD14克隆M5E2(PE-Cy7)(例如Becton Dickinson ,目录号:557742 ),4 °C

CD3克隆UCHT1(BV421)(例如Becton Dickinson ,目录号:562426 ),4 °C

CD19克隆SJ25C1(BV510)(Becton Dickinson公司,例如,目录号:562 947 ),4 ℃下

MACS缓冲液(请参见配方),4 °C

稀释介质(参见配方),4 °C

洗涤介质(请参见食谱),4 °C

 

仅对于iPSC衍生的NK细胞分离是必需的

30 µm CellTrics过滤器(Sysmex,目录号:04-004-2326 )

CD7克隆124-1D1(eBioscience,例如,目录号:25-0079-41 ),4°C或克隆4H9(例如Becton Dickinson ,例如,目录号:347483 ),4°C或克隆M-T701(Becton Dickinson ,目录号:561934 ),4 °C

 

刺激性

实验室一次性用品:

P ipett ES

15毫升管

50毫升管

Ë ppendorf恩BES

96 -孔圆底板(格雷纳,目录号:650185)

AIMV介质(Thermo Fisher Scientific,Gibco,目录号:31035025)

胎牛血清(FCS,在56 °C加热30分钟以灭活补体)(Merck,Sigma - Aldrich,目录号:F7524 ),- 20°C

重组人IL-12(Peprotech,目录号:200-12 ),-20 °C

重组人IL-15(Peprotech,目录号:200-15 ),-20 °C

重组人IL-18(MBL International,目录号:B001-5 ),-20 °C

白介素混合物(请参阅食谱)

 

功能分析

实验室一次性用品:

P ipettes

15 ml聚丙烯管

0.5 ml聚丙烯管

[R eagent水库

铝箔

密封胶带(例如,Merck,Grei ner,目录号:A5596-100EA)



纸巾

Bio-Plex Pro人类细胞因子27重免疫分析法(Bio-Rad,目录号:M5 0-0KCAF0Y ),4 °C

 

设备

 

用于无菌工作的层流柜(生物安全等级II)(Euroflow EF4,Baker的CleanAir)

可容纳15个50毫升试管的试管架(例如,VWR,目录号:89215-778和89215-778)

微量移液器(P10,P100,P1000)(例如,Gilson,目录号:F167380 )

12通道多通道移液器(例如,Eppendorf,目录号:3125000060 )

台式离心机,带有用于板以及15 ml和1.5 ml管的适配器(Eppendorf,型号:5810 R)

CO 2培养箱(在5%CO 2和37°C下)(例如,Panasonic,型号:MCO 170-AIC)

电磁搅拌器(例如,VWR,目录号:89215-778)

FACS Aria细胞分选仪(Becton Dickinson ,Aria I,II,III),但任何等效的荧光激活细胞分选仪都可以使用

MiniMACS或MidiMACS分隔器(Miltenyi,目录号分别为130-042-102或130-042-302)

MACS MultiStand(Miltenyi,货号:130-042-303,RT)

高压灭菌器(例如,VWR,病房,目录号:470230-598)             

Vortex(例如Scientific Industries,型号:Vortex Genie 2,目录号:200-SI-0236)

Bio-Plex 200系统(例如Bio-Rad ,目录号:171000201)

Bio-Plex手持式电磁清洗机(Bio-Rad,目录号:171020100)

平板振荡器(例如,Biosan,型号:PSU-2T)

 

软件

 

Diva软件(Becton Dickinson,v6.0或更高版本,2007或更高版本)

Bio-Plex Manager软件(Bio-Rad,v6.2,2018)

 

程序

 

原代NK细胞分离

A.外周血单核细胞(PBMC)分离      

注意:除非另有说明,否则为室温(RT)。

稀至少15毫升血液在2倍稀释介质并混合。

注意:通常可以从15毫升血液中分离出约1.5 x10 6个NK细胞。该数目将产生〜7.5 x 10 4个CD56亮NK细胞。

将4 ml Ficoll加入15 ml管中。

小心地将稀释的血液样本(10毫升)铺在Ficoll上(不要混合)。

重复小号TEP小号2和3为剩余稀释的血液。

离心15分钟,在1 ,000 ×g下无制动。

通过小心吸取(使用巴斯德吸管)血浆和聚蔗糖之间的白色细胞层来收获PBMC。

将PBMC转移到新的50 ml试管中(每个样品池最多25 ml)。

加入最多50毫升的洗涤介质。

以800 xg离心10分钟。

抽吸除去上清液并重悬细胞。

如果适用,将细胞倒入150 ml管中。

加入最多10毫升的洗涤介质。

离心10分钟,在540 ×g下。

除去上清液,然后将细胞重悬在建议体积的MACS缓冲液中(每10毫升未稀释的血液约1毫升MACS缓冲液)。

用Türk溶液计数细胞(用90 µlTürk溶液稀释10 µl细胞悬液)。将其余电池保持在4°C。

保持2× 10 6 PBMC静置单染色CON trols在索尔蒂纳克过程。

 

B.使用MACS从PBMC分离NK细胞      

注意:在NK细胞分离过程中,将细胞和缓冲液保持在4°C 。查看制造商的说明,以了解协议中的任何更新。

将细胞转移到15 ml试管中,并加入14 ml MACS缓冲液。

离心5分钟540 xg 。

注意:磁性标记的体积是最小体积,当使用少于1 x 10 7的单元格时,请使用此体积,当使用超过1 x 10 7的单元格时,请相应地按比例放大体积。

抽吸除去上清液。每1 x 10 7个细胞在40 µl MACS缓冲液中重悬细胞。

示例:F或1.5 x 10 7个细胞,添加60 µl MACS缓冲液

每1 x 10 7个细胞添加10μlNK细胞生物素-抗体混合物。

充分混合并在4 °C下孵育5分钟。

每1 x 10 7个细胞添加30 µl MACS缓冲液。

每1 x 10 7个细胞添加20 µl NK细胞MicroBead鸡尾酒。

充分混合并在4 °C下孵育10分钟。

将色谱柱(MS≤1 x 10 7池,LS≥1 x 10 7 PBMC)放在MACS分离器的磁场中,该磁场位于MACS MultiStand上。每个LS列最多可加载1 x 10 8 PBMC。

将预分离过滤器放在色谱柱顶部。

将色谱柱放入15 ml管中。

通过移液器中的MACS缓冲液冲洗色谱柱:500 µl(MS色谱柱),3 ml(LS色谱柱)

用新的15 ml管替换装有缓冲液的15 ml管(带有NK细胞的流通液将收集在该管中)。

重要提示:Ø NLY吸管新卷当列水库是空的。

用移液器吸取标记的PBMC。磁分离最小体积为500 µl。如有必要,将MACS缓冲液添加到细胞悬液中。

通过吸取过滤器中的MACS缓冲液来洗涤色谱柱:500 µl (MS色谱柱),3 ml(LS色谱柱)

重复小号TEP 15的两倍。

将装有未标记NK细胞的15 ml离心管离心,并以540 xg离心5分钟。

重悬于MACS缓冲液(〜500-1 ,000微升)和COUN吨。

 

C. NK细胞亚群的FACS      

注意:将细胞和缓冲液保持在4 °C 。

Pr epare抗体混合物(每1 x 10 6 NK细胞30 µl )。通过保持抗体混合物在黑暗中来防止漂白!见ř ecipes例如计算。重要提示:使用滴定的抗体。

注意:使用推荐的稀释液作为5个浓度的中位数,对10 6 PBMC进行滴定。通常应用2倍稀释系数。建议的稀释液应达到饱和,但同时不得改变抗原阴性细胞的平均荧光强度。

制备单一污渍对照混合物(1种抗体+ MACS缓冲液),每种单一污渍25 µl。使用与完整抗体混合物相同的稀释度。不要忘记包括1个未染色的对照。

将2 x 10 5 PBMC转移到96孔板的一个孔中(1孔/单个污渍)。

离心沉淀细胞5分钟,540 ×g下在4 ℃下(对于管),或2分钟,540 ×g下在4 ℃下(对于板)。

对于试管:通过抽吸除去15 ml管中的上清液,并以正确量的抗体混合物重悬。在4°C下于黑暗中孵育30分钟。

对于板:轻扫水槽上方的板,以除去孔中的上清液。轻轻将平板轻拍一次在纸巾上,以除去所有清洁液。加入25 µl单一污渍混合物。在4 °C下于黑暗中孵育30分钟。

通过向细胞中加入10 ml (用于试管)和200 µl (用于板)MACS缓冲液来洗涤细胞。

离心沉淀细胞如前:5分钟,540 ×g下在4℃下(对于管),2分钟,540 ×g下在4℃下(对于板)。

如前描述(除去上清液小号TEP 5)。

对于试管:将NK细胞重悬于MACS缓冲液中,使其浓度为每毫升1 x 10 7 NK细胞。

对于板:将PBMC重悬于70 µl MACS缓冲液中,并将其收集在微管中。

用3 ml收集培养基制备圆底聚苯乙烯试管(最多可在1个试管中收集6 x 10 5个细胞)。

排序CEL在低压LS(100微米的喷嘴,20 PSI,这是对NK细胞的分选后的功能是重要的)。如图1所示。如果更多的子集需要,CD56使用门控策略明亮可以进一步细分在CD16 -和CD16 +子集。1.5 x 10 6 NK细胞的分选将产生(在考虑细胞损失的情况下)〜7.5 x 10 4 CD56亮NK细胞和1 x 10 6 CD56暗色NK细胞。

 




图1.用于分类原代NK细胞亚群的门控策略。首先,根据前向散射和侧向散射对淋巴细胞进行门控。接着,双峰,CD3 + T细胞,CD19 + B细胞,CD14 +单核细胞和CD33 + CD56 -髓样细胞被排除在外。NK细胞可根据CD56和CD16的强度表达进一步细分:CD56亮CD16 +/-和CD56暗CD16 + NK细胞。

 

通过FACS分离iPSC衍生的NK细胞

注意:在NK细胞分离过程中,将细胞和缓冲液保持在4°C 。

将iPSC分化培养物中的漂浮细胞(图2A)收集到15 ml或50 ml锥形管中。管的尺寸取决于分化的规模(典型格式:从6几个孔-孔平板)。

注意:通常从1个6孔板中收获约1 x 10 6个浮细胞。根据分化的成功程度,有20-70%的漂浮细胞是CD7 + CD56 + NK细胞。

使细胞悬液通过30 µm CellTrics过滤器(Sysmex),并在15 ml或50 ml锥形管中收集细胞。

在4°C下以400 xg离心收集的细胞悬液5分钟。

卸下supernatan通过抽吸吨重悬沉淀的细胞在500微升冰冷的PBS(洗涤小号TEP 1)。

在4°C下以400 xg离心5分钟。

通过抽吸除去上清,重悬在500沉淀的细胞微升冰冷的MACS缓冲液(洗涤小号TEP 2)。

在4°C下以400 xg离心5分钟。

通过抽吸除去上清液的d重悬在500沉淀的细胞微升冰冷的MACS缓冲液(洗涤小号TEP 3)。

取一小份(10微升)并计数细胞。

将细胞转移到0.5 ml Eppendorf管或96孔板中(圆底或V形底部孔)。所需的试管/孔数:未染色的对照,单一染色对照,完全染色。使用2× 10个4 -5× 10 4 Ç厄尔为单染色或未染色的对照。其余的用于将要分类的单元格的组合染色。切勿使用1 x 10 6细胞/ 25 µl染色液。

在4°C(用于试管)中以400 xg离心5分钟,或在4°C(用于板)中以540 xg离心2分钟。

准备抗体混合物:单一染色质对照混合物(1个抗体+ MACS缓冲液),每个单一染色质25 µl,以及至少包含抗CD7和抗CD56的完整抗体混合物,以鉴定NK细胞样细胞。

注意:对于相同的抗体,请使用相同的稀释液,并使抗体避光,尤其是在将串联染料用作缀合物时。别忘了包括一个未染色的对照,最好还包括一个由单个核血细胞组成的阳性对照,例如外周血。

通过抽吸(用于试管)或在水槽上方轻拂板(用于板)除去上清液。轻轻将平板轻拍一次在纸巾上(用于平板),以除去所有清洁液。

重悬抗体混合物中的细胞。

在4°C下(通常在冰箱中)在黑暗中孵育30分钟。

在4°C(用于试管)中以400 xg离心5分钟,或在4°C(用于板)中以540 xg离心2分钟。

如前所述除去上清液(第13步),并将细胞重悬于200 µl冰冷的MACS缓冲液中。

在4°C(用于试管)中以400 xg离心5分钟,或在4°C(用于板)中以540 xg离心2分钟。

如前所述除去上清液(第13步),并将单个染色剂,未染色的细胞和阳性对照细胞重悬于70 µl MACS缓冲液中,并将其转移至Micronic或5 ml圆底聚苯乙烯管中。将要分选的细胞以每毫升10 x 10 6个细胞的浓度重悬。

用3 ml收集培养基准备5 ml圆底聚苯乙烯试管(最多可在1个试管中收集6 x10 5个细胞)。

在低压(100 µm喷嘴,20 PSI)下对CD7 + CD56 +细胞进行分选。使用如图2B所示的门控策略。

注意:根据要解决的问题,可以使用针对其他NK细胞标记(例如CD16)的所需纯度抗体。

 




图2.通过FACS分离iPSC衍生的NK细胞。一。与OP9-DL1细胞共培养3周(放大100倍)后,iPSC向NK细胞分化的一个例子。收集非贴壁细胞,并使用FACS分离CD56 + NK细胞。乙。隔离iPSC衍生的CD7 + CD56 + NK细胞的门控策略。

 

NK细胞刺激

1.旋转分选的NK细胞亚群,以5.6 x 10 4细胞/ ml的浓度重悬于含1%青霉素-链霉素的AIMV培养基中(最终FCS浓度为5%)。        

2.在无菌的96孔圆底平板中加入180 µl /孔NK细胞。        

3.在不同的孔中加入20 µl IL-12,IL-15和IL-18混合物以及不含细胞因子的培养基(AIMV + 1%青霉素-链霉菌素)。        

注意:在IL-12,IL-15和IL-18中培养的健康供体细胞是IFN-γ产生的阳性对照。在没有细胞因子的培养基中培养的细胞充当阴性对照。优选地,应一式三份进行刺激。如果分拣的细胞超过了所需的单细胞因子或双细胞因子混合物刺激,则应包括在内。

4.包括1孔200 ul培养基(AIMV + 1%青霉素-链霉菌素+ 5%FCS)和1孔200 ul补充白介素的培养基(作为Luminex的背景)。        

5.用塑料箔纸包住板以防止蒸发。        

6.在37°C,5%CO 2的培养箱中培养细胞20小时。        

注意:研究动力学时,请在多个时间点取样10 µl。

7.旋转540 xg的平板2分钟。        

8.收集上清液,并保存在-20 °C的密封96孔板或Micronic管中。        

 

Luminex

注意:以下协议已进行优化,因此与制造商的说明不同。

降速的标准小瓶中,并重建标准的单个小瓶中加入500μl稀释液(=培养基AIMV + 5%FCS),轻轻涡旋5秒并在冰上孵育30分钟。

标记9个0.5 ml聚丙烯管Std1-Std8和空白。

在每个试管中添加指定体积的稀释剂(表1)。

轻轻涡旋重组标准液5秒钟,取128 µl并添加到Std1管中。

涡旋Std1并将50 µl从试管Std1转移到试管Std2并涡旋。重要说明:每次进行体积传输时,请使用新的移液器吸头。

通过每次转移50 µl,继续进行S2至S8管的系列稀释。




表1.连续稀释以生成标准曲线



稀释剂(= AIMV + 5%FCS)

转移量

标准1

72微升

128 µl标准液

标准2

150微升

50 µl Std1

标准3

150微升

50 µl标准液2

标准4

150微升

50 µl Std3

标准5

150微升

50 µl Std4

标准6

150微升

50 µl Std5

标准7

150微升

50 µl标准6

标准8

150微升

50 µl标准7

空白

150微升

--

 

解冻的上清液,并离心以1 ,000 ×g下在室温下4分钟。

将上清液转移到干净的聚丙烯管中。

在稀释前将样品以1:6的比例稀释(融化后将其置于冰上)并平衡至室温。

进行板布局(例如图3),以检查所需的孔数。使用标准品和空白品的副本。垂直填充板。

 




图3.印版布局。带有8个标准品和1个空白样品(一式两份)和6个样品的板布局示例。

 

将所需体积的测定缓冲液添加到15 ml管中(表2)。

以中等速度涡旋广告30秒。小心地打开瓶盖,然后将残留在瓶盖中的所有液体吸回小瓶中。重要提示:请勿离心原液珠,因为这些珠会被甩下来。

通过吸取所需稀释珠的股票珠体积到15ml试管(Ť能够2)。重要提示:防止光珠与一个luminium箔。




表2.珠稀释液

韦尔斯

10x股票珠

测定缓冲液

总容积

96

288微升

5,712微升

6,000微升

48

144微升

2,856微升

3,000微升

 

使用前将标准品和样品带入RT。

将稀释的微珠以中等速度涡旋振荡30 s,然后将微珠倒入试剂容器中。将50 µg珠子转移到平板底部的每个孔中。

每孔加入100μlh缓冲液。将平板放在手持磁铁上至少60 s,然后快速倒出废液。将平板轻拍到纸巾上以除去所有检修液。从磁铁上取下磁碟,轻敲磁碟,小心地重悬磁珠,然后重复此洗涤步骤。

轻轻涡旋稀释的标准液,空白样品和样品5秒钟。转移50微升到每个孔。重要:每次进行体积传输时,请使用新的移液器吸头。

用新的密封胶带覆盖板子,并用铝箔纸避光。在RT上于450 rpm的振荡器上孵育45分钟。

使用前准备10分钟稀释检测抗体。将所需体积的检测抗体稀释液添加到新的15 ml管中(表3)。

以中等速度涡旋原种检测抗体15-20 s。降低库存30秒钟。

通过将所需体积移液到15 ml管中来稀释10x检测抗体(表3)。

孵育45分钟后,取下密封胶带,并用100 µl洗涤缓冲液洗涤3次。

将稀释的检测抗体涡旋振荡5 s ,倒入试剂槽中,并向每个孔中转移12.5 µl。

用新的密封胶带盖好盖板,并用铝箔纸避光。在RT上于450 rpm的振荡器上孵育30分钟。

 

表3.检测抗体的稀释

韦尔斯

10x检测抗体

检测抗体稀释液

总容积

96

150微升

1,350微升

1,500微升

48

75微升

675微升

750微升

 

使用前,准备10分钟的链霉亲和素-PE稀释液。将所需体积的测定缓冲液添加到新的15 ml管中(表4)。

 

 

 

表4.稀释链霉亲和素-PE

韦尔斯

100倍SA-PE

测定缓冲液

总容积

96

30微升

2,970微升

3,000微升

48

15微升

1,480微升

1,500微升

 

以中等速度涡旋100x SA-PE 5 s,然后旋转30 s。

移取所需体积的SA-PE到15 ml管中(表4)。涡旋并避光。

接通Bio-plex系统的电源(提前30分钟)。

孵育30分钟后,取下密封胶带,并用100 µl洗涤缓冲液洗涤平板3次。

将稀释的SA-PE涡旋振荡5 s,倒入试剂槽中,并向每个孔中转移25 µl。

用密封胶带和铝箔覆盖板。在RT上于450 rpm的振荡器上孵育10分钟。

用100 µl洗涤缓冲液洗涤平板3次。

要重悬磁珠以读取板,请在每个孔中加入80 µl分析缓冲液,并用密封胶带覆盖板。在RT上于850±50 rpm的振荡器上孵育30 s。

撕下胶带并阅读印版(使用较低的PMT设置)。如果您希望浓度较低,请使用较高的PMT设置。

数据分析:对于一个详细的教程,我们Bio-Rad公司(参见在线教程[R eference 1为实例)。

注意:它以减去背景值从其它值,其通过基于标准曲线上的软件自动生成(用补充有白细胞介素只中等或中等孔)是重要的。此外,通过验证阴性对照(无刺激的细胞)确实是阴性,以及阳性对照(受IL-12,IL-15和IL-18刺激的细胞)是否显示IFN-γ来检查实验是否成功。 。图4显示了各种NK细胞产生细胞因子的例子。

 




图4. Luminex测量刺激的原代和iPSC衍生NK细胞的实例。Bio-Plex Pro人类细胞因子27重免疫分析法可检测27种细胞因子/趋化因子。NK细胞主要是GM-CSF ,TNF-α和IFN-γ以及各种趋化因子的产生者。GM-CSF ,TNF-α和IFN-γ生产中示出在初级的培养NK细胞亚群(CD56明亮和CD56暗淡)和源自iPSC的NK细胞(CD7 + CD56 +中IL-12,IL的存在下) -15和IL-18 20小时。数据代表至少2个独立实验。显示了平均值和标准偏差。

 

菜谱

 

稀释介质

500 ml RPMI 1640培养基

5 ml青霉素-链霉素100x

将青霉素-链霉素添加到RPMI 1640培养基中并摇匀

洗涤介质

500 ml RPMI 1640培养基

5 ml青霉素-链霉素100x

2毫升人血清白蛋白200克/升(最终= 0.8克/升)

将青霉素-链霉素和人血清白蛋白加入RPMI 1640培养基中并摇匀

EDTA溶液0.5 M

1000毫升蒸馏水

186.12克EDTA

将EDTA加入装有蒸馏水的瓶子中,添加磁力搅拌棒,然后将瓶子放在磁力搅拌器上。加入沉淀的NaOH,直到EDTA溶解(pH 7.5-8左右)。高压灭菌,并将瓶装在室温下。使用注射器过滤0.22 μ米过滤使用前将溶液

MACS缓冲区

500毫升PBS

8.3毫升30%BSA(最终= 0.5%)

2 ml 0.5 M EDTA溶液(最终= 2 mM)

将BSA和已过滤的EDTA溶液添加到PBS中并混合

原代NK细胞的抗体混合物

MACS缓冲区

1:20 CD33 PE

1:200 CD16 FITC

1:100 CD56 ECD

1:100 CD14 PE-Cy7

1:200 CD3 BV421

1:100 CD19 BV510

将抗体加入MACS缓冲液(在Eppendorf管中)并涡旋。保持混合物在黑暗中于4 °C

收集介质

AIMV培养基

30%FCS

1%青霉素链霉素

将FCS和青霉素-链霉素添加到AIMV培养基中并混合

白介素混合物

AIMV培养基

1%青霉素-链霉素

10 ng / ml IL-12

10 ng / ml IL-15

20 ng / ml IL-18

将青霉素-链霉素和白介素加入AIMV培养基并涡旋

 

致谢

 

这项研究得到了NWO(JM),莱顿大学医学中心奖学金(JM),LSBR基金会(HM)(LSBR09-11),欧洲委员会(玛丽·居里MT奖学金),荷兰癌症协会的研究生计划的支持。 (KWF)(MT)和Stichting VUmc CCA(MT)。

 

利益争夺

 

所有作者都宣称没有利益冲突。

 

伦理

 

从健康成人供体的血沉棕黄层(Sanquin血库,西南地区,鹿特丹,荷兰)中分离出原代NK细胞。iPSC的人类材料是根据Erasmus MC(MEC-2016-606)或LUMC(P13-080)的“医学伦理委员会”的批准收集的。涉及人体材料的实验是根据“赫尔辛基宣言”中概述的原则进行的。

 

参考文献

 

Bio-Plex Manager TM和Data Pro TM软件:简化多重数据分析(2016 )。YouTube ,2月1日https://www.youtube.com/watch?v=v2i3Vp6MwyE。

Eberlein,J.,Nguyen,TT,Victorino,F.,Golden-Mason,L.,Rosen,HR和Homann,D.(2010)。通过流式细胞仪全面评估趋化因子表达谱。J临床投资120(3):907-923。

D.Knorr,Ni,Z.,Hermanson,D.,Hexum,MK,Bendzick,L.,Cooper,LJ,Lee,DA和Kaufman,DS(2013)。从人类多能干细胞用于癌症治疗的自然杀伤细胞的临床规模衍生。干细胞Transl Med 2(4):274-283。

Lugthart,G.,Melsen,JE,Vervat,C.,van Ostaijen-Ten Dam,MM,Corver,WE,Roelen,DL,van Bergen,J.,van Tol,MJ,Lankester,AC和Schilham,MW(2016 )。人淋巴组织具有不同的CD69 + CXCR6 + NK细胞群体。免疫学杂志197(1):78-84。

梅尔森(JE),卢格塔特(G.Lugthart),AC兰开斯特(Lankester)和西蒙纳瓦州席勒姆(Shilham)(2016)。人类循环和组织驻留CD56明亮的自然杀伤细胞群体。正面免疫7:262。

米特里(M.),卡塔(A.),博斯马(A.),普林斯(Prins),约翰逊(J.),科德斯(C。),M。,德威尔特(B.),法拉哈尼(F. Staal,FJT和Mikkers,HMM(2020)。基于iPSC的RAG2严重联合免疫功能低下的模型揭示了多个T细胞发育停滞。干细胞报告14(2):300-311。

M.Themeli,M.Kloss,CC.Ciriello,M.,Fedorov,VD,Perna,F.,M.Gonen和M.Sadeelain(2013)。从诱导的多能干细胞产生靶向肿瘤的人T淋巴细胞用于癌症治疗。Nat Biotechnol 31(10):928-933。

曾俊杰,唐SY,Toh LL和Wang S.(2017)。从外周血细胞衍生的多能干细胞中产生“现成的”自然杀伤细胞。干细胞报告9:1796-1812。
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引用:Melsen, J., Themeli, M., van Ostaijen-ten Dam, M., van Beelen, E., Lugthart, G., Hoeben, R., Schilham, M. and Mikkers, H. (2020). Protocol for Isolation, Stimulation and Functional Profiling of Primary and iPSC-derived Human NK Cells. Bio-protocol 10(23): e3845. DOI: 10.21769/BioProtoc.3845.
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