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Jan 2016

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Adoptive Transfer of Monocytes Sorted from Bone Marrow
骨髓源性单核细胞的过继转移   

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Abstract

Inflammatory Ly6Chi monocytes can give rise to distinct mononuclear myeloid cells in the tumor microenvironment, such as monocytic myeloid-derived suppressor cells (Mo-MDSC), immature macrophages, M2-like tumor-associated macrophages (TAMs), M1-like TAMs or monocyte-derived dendritic cells (Mo-DCs). This protocol describes a method to assess the fate and recruitment of inflammatory Ly6Chi monocytes in the tumor microenvironment.

Keywords: Adoptive transfer (过继转移), Monocytes (单核细胞), Pre-cDCs (Pre-cDCs), Tumor-associated macrophages (肿瘤相关巨噬细胞), Tumor-associated dendritic cells (肿瘤相关树突状细胞), Cell sorting (细胞分选)

Background

Tumors are heterogeneous microenvironments where complex interactions take place between neoplastic cells and infiltrating inflammatory cells, such as tumor-associated macrophages (TAMs) and tumor-associated dendritic cells (TADCs). The relevance of tumor-infiltrating mononuclear myeloid cells is underscored by clinical studies showing a correlation between their abundance and poor prognosis (Bolli et al., 2007). The origin of TAMs and TADCs has been a matter of debate, since several levels of complexity result in considerable TAM and TADC heterogeneity (Movahedi et al., 2010; Laoui et al., 2014, Laoui et al., 2016; Van Overmeire et al., 2016; Kiss et al., 2018). Here, we describe a valuable method to adoptively transfer bone-marrow derived monocytes permitting the assessment of their recruitment and fate in tumors.

Materials and Reagents

  1. Polyester filters cut in 10 x 10 cm squares, thread diameter 70 μm (Specturmlabs, catalog number: 146490)
  2. 10 ml syringes (Omnifix, catalog number: 473203)
  3. 1 ml syringes (Greiner, catalog number: 470203)
  4. 27 G needles (BD Bioscience, catalog number: 300635)
  5. 25 G needles (BD Biosciences, catalog number: 300400)
  6. 19 G needles (BD Biosciences, catalog number: 301500)
  7. Falcon standard tissue culture dish (Fisher Scientific, catalog number: 353003)
  8. BD Falcon 50 ml polypropylene tubes (BD Biosciences, catalog number: 2070)
  9. BD Falcon 15 ml polypropylene tubes (BD Biosciences, catalog number: 2096)
  10. BD Falcon 5 ml polypropylene round-bottom tube (BD Biosciences, catalog number: 352063)
  11. 70 µm sterile nylon gauze
  12. LS columns (Miltenyi, catalog number: 130-042-401)
  13. Naive mice: Age preferably between 6 and 12 weeks, strain can vary depending on the experiment/project (in this example we used C57BL/6 mice)
  14. Ethanol absolute analaR Normapur ACS (VWR Chemicals, catalog number: 84857360)
  15. RPMI-1640 medium (RPMI) (Life Technologies, catalog number: 52400-041)
  16. Fetal calf serum (FCS) (Life Technologies, Gibco, catalog number: DE14-801F)
  17. L-glutamine (Life Technologies, catalog number: 25030-024)
  18. Penicillin-streptomycin (Life Technologies, catalog number: 15140-130)
  19. Ammonium chloride (NH4Cl) (Merck KGaA, catalog number: 1011450500)
  20. Potassium bicarbonate (KHCO3) (Merck KGaA, catalog number: 104852)
  21. EDTA (Duchefa Biochemie, catalog number: E0511.1000)
  22. Hank’s buffered salt solution (HBSS) (Life Technologies, Gibco, catalog number: 14175129)
  23. Anti-CD11b microbeads (Miltenyi, catalog number: 130-049-601)
  24. Purified CD16/CD32 (FcBlock) (clone 2.4G2) (BD Biosciences, catalog number: 553142)
  25. PE-Cy7-conjugated anti-CD11b antibody (clone M1/70) (BD Biosciences, catalog number: 552850) 
  26. AF647-conjugated anti-Ly6C antibody (clone ER-MP20) (Serotec, catalog number: MCA2389A647) 
  27. PerCP-Cy5.5-conjugated anti-I-A/I-E (MHC-II) antibody (clone M5/114.15.2) (BioLegend, catalog number: 107626) 
  28. FITC-conjugated anti-Ly6G antibody (clone 1A8) (BD Biosciences, catalog number: 551460) 
  29. APC-Cy7-conjugated anti CD45 (clone 30-F11) (BioLegend, catalog number: 103116) 
  30. CellTrace Violet (Thermo Fisher Scientific, Molecular probesTM, catalog number: C34557)
  31. Trypan blue (Life Technologies, Gibco, catalog number: 15250061)
  32. DMSO
  33. 70% ethanol (see Recipes)
  34. Complete medium (see Recipes)
  35. Erythrocyte lysis buffer (see Recipes)
  36. MACS buffer (see Recipes)
  37. Sorting buffer (see Recipes)
  38. Violet tracer (see Recipes)

Equipment

  1. Sterile culture hood, PSM Optimale 18 (ADS)
  2. Surgical scissors and forceps
  3. 37 °C, 5% CO2 cell culture incubator (Binder, VWR)
  4. Pipettes (Gilson)
  5. Centrifuges 5810 R (Eppendorf, model: 5810 R)
  6. Shaker KS 260 Basic (IKA, model: KS 260 basic)
  7. Microscope Eclipse TS100 (Nikon, model: Eclipse TS100)
  8. MidiMACSTM Separator and MultiStand (Miltenyi, catalog number: 130-042-301)
  9. Multicolor FACS Sorter-FACS Aria II (BD Biosciences Aria flow cytometer)

Procedure

  1. Preparation of a bone-marrow single cell suspension
    1. Sacrifice a naive mouse and restrain it by pinning its paws into a foam surface using syringe needles. Disinfect the skin of the mouse with 70% ethanol (see Recipe 1). Make a parallel incision from the base of the tail up to the neck along the mouse’s abdomen and to the paws without puncturing the peritoneum. Gently pull back the skin and pin it to the foam surface to expose the hind limb (Figure 1A).
    2. Cut the hind limb free from the skin and the body by cutting in the pelvis just behind the femur-pelvis joint. Keep the femur and tibia whole. Try to remove as much excess tissue (muscles, fibers...) surrounding the bone as possible using scissors or with a scalpel (Figure 1B). Do this procedure gently in order to avoid breakage of the bones. 
    3. Gently pull the hind paw from the limb by moving it back and forwards (Figure 1C). 
    4. Clean the bone by submerging it in 70% ethanol and store the bone in 5 ml complete medium (see Recipe 2) in a 50 ml Falcon tube on ice.
    5. Repeat this action with the second hind limb.
    6. Detach the tibia from the femur and cut the fibula and patella away and put the bones in a Falcon standard tissue culture dish (Figures 1D, 1E and 1F).
    7. Insert a 27 G needle attached to a 10 ml syringe containing complete medium in the femur and gently flush the femur with 10 ml complete medium (Figures 1G and 1H). Before flushing, the tibia cut the ‘white’ bone that was adjacent to the paw away and insert the needle in the bone at the other side. If any resistance is perceived when flushing the bones, cut a small fraction of the bone from the end and reinsert the needle in the remaining bone. The flushing is complete when all the red bone marrow is in the plate, and red tissue can no longer be seen in the bone.
    8. Homogenize the bone-marrow by passing (aspirating and pressing) the medium containing bone-marrow two times through the 19 G needle (Figures 1I and 1J).
    9. Filter the bone-marrow suspensions through a 70 µm sterile nylon gauze into a sterile 50 ml conical tube and wash the culture plate and the gauze with 10 ml complete medium.
    10. Centrifuge the 50 ml tubes at 450 x g for 6 min at 4 °C and discard the supernatants (Figure 1K).
    11. Remove the red blood cells by resuspending the pellet in 5 ml erythrocyte lysis buffer (see Recipe 3) and leave at room temperature for 2 min. 
    12. Neutralize by adding 15 ml complete medium, and transfer the suspension to a new 50 ml tube through a 70 µm sterile nylon gauze. 
    13. Centrifuge the 50 ml tubes at 450 x g for 6 min at 4 °C and discard the supernatants (Figure 1L).
    14. Count the living cells using trypan blue and resuspend the cells in MACS buffer (see Recipe 4) at a concentration of 108 cells/ml. From the total bone marrow, in general, 10% to 15 % normally would constitute monocytes (varies slightly with age, mouse strain, and animal house type), which can be enriched/purified as explained below.


      Figure 1. Bone-marrow single-cell preparation. A. Naive mouse. B. Hind limb with paw. C. Hind limb without paw. D. Cleaned hind limb without paw. E. Detaching of the tibia from the femur. F. Femur (left) and tibia (right). G. Flushing the bones. H. Bone marrow right after flushing. I. Homogenization of the bone marrow. J. Homogenized bone-marrow single cell suspension. K. Cell pellet before erythrocyte lysis buffer. L. Cell pellet after erythrocyte lysis buffer.

  2. Purification of Ly6Chigh monocytes from the bone marrow
    1. Add a 5 µl aliquot of anti-CD11b magnetic microbeads per 107 cells and incubate for 20 min at 4 °C on an orbital shaker at 50 rpm.
    2. Wash by adding 10 ml MACS buffer, centrifuge at 450 x g for 6 min at 4 °C and discard the supernatants.
    3. Place an LS column in a MidiMACSTM Separator attached to a magnetic MultiStand and wash it by putting 3 ml MACS buffer on the top. The liquid passes the column by gravity.
    4. Resuspend the pelleted cells in 1 ml MACS buffer and pipette the labeled cell suspension on top of the LS separation column. When the cell suspension has passed through the column, wash the column by adding 3 x 3 ml MACS buffer.
    5. Remove the LS column from the separator, add 5 ml MACS buffer on top of the column and immediately flush the column by firmly pressing the provided plunger on the column to wash the magnetically labeled cells out in a sterile 15 ml tube.
    6. Incubate the CD11b+ cell suspension with rat anti-mouse CD16/CD32 (10 µg per 107 cells) on ice-cold water for 20 min, in order to block the Fc receptors present on the cells’ surface.
    7. Incubate the cell suspension with fluorescently labeled antibodies of interest (1 µg per 107 cells) for another 20 min on ice-cold water, protected from exposure to light. 
    8. Wash by adding 10 ml MACS buffer, centrifuge at 450 x g for 6 min at 4 °C and discard the supernatants.
    9. Meanwhile, precoat 5 ml polypropylene round-bottom tubes and 15 ml tubes with heat-inactivated fetal calf serum, add respectively 1 ml or 2 ml heat-inactivated fetal calf serum. Shake the tubes gently by hand so that the heat-inactivated fetal calf serum covers the whole surface of the tube, and discard the excess of heat-inactivated fetal calf serum. This will prevent the cells to stick to the tubes and hence enhance the recovery of cells.
    10. Resuspend the pellet in 1 ml sorting buffer (see Recipe 5) per 107 cells and transfer into a sterile 5 ml polypropylene round-bottom tube precoated with heat-inactivated fetal calf serum.
    11. Ly6Chigh monocytes can be sorted on a BD FACS Aria as CD45pos CD11bpos Ly6Gneg Ly6Chigh MHC-IIneg cells (Laoui et al., 2016; Van Overmeire et al., 2016).
    12. Collect the sorted monocytes in 15 ml tubes precoated with heat-inactivated fetal calf serum containing 3 ml complete medium.

  3. Labeling of the monocytes for in vivo tracking
    1. Option I: Bone-marrow donor mice are wild-type mice (Laoui et al., 2016). Ideally, the donor and recipient differ in their CD45 allele (CD45.1 vs. CD45.2).
      1. Centrifuge the 15 ml tubes containing the sorted monocytes at 450 x g for 10 min at 4 °C and discard the supernatant.
      2. Resuspend the cell pellet in HBSS at a concentration of 106/ml and add 1 μl of CellTrace (see Recipe 6) to stain 1 ml cells (hence a 1:1,000 dilution). It is important that the labeling happens in a protein-free medium. Incubate the cell suspension for 20 min at 37 °C, protected from exposure to light.
      3. Wash by adding 10 ml HBSS, centrifuge at 450 x g for 6 min at 4 °C and discard the supernatant.
      4. Resuspend the cell pellet in HBSS at a concentration of 5 x 106/ml and keep on ice till entering the animal facility.
      5. Inject the recipient CD45.2 mice with 200 μl intravenously in the tail vein using a 25 G needle and a 1 ml syringe (Video 1).

        Video 1. Intravenously tail vein injection. (This video was made at Vrije Universiteit Brussels according to guidelines from the Belgian Council for Laboratory Animal Sciences and were approved by the Ethical Committee for Animal Experiments of the Vrije universiteit Brussels (license 15-220-2).

      6. The progeny of the monocytes can be traced as from one day, until 10 days after inoculation (ideally 48 to 72 h). The monocyte-derived cells can be traced in the Pacific Blue-channel (405 nm) by flow cytometry. In addition, CD45.1 and CD45.2 can be used as a complementary staining.

    2. Option II: Bone-marrow donor mice are Ubiquitin-GFP mice (Van Overmeire et al., 2016).
      1. Centrifuge the 15 ml tubes containing the sorted monocytes at 450 x g for 10 min at 4 °C and discard the supernatant.
      2. Resuspend the cell pellet in HBSS at a concentration of 5 x 106/ml and keep on ice till entering the animal facility.
      3. Inject the recipient CD45.2 mice with 200 μl of cells intravenously in the tail vein using a 25 G needle and a 1 ml syringe (Video 1).
      4. The progeny of the monocytes can be traced as from one day, until 10 days after inoculation (ideally 48 to 72 h). The monocyte-derived cells can be traced in the FITC-channel by flow cytometry.

Data analysis

Flow cytometry is commonly used to visualize the transferred monocytes in the tumors of the recipient mice. As the transferred monocytes are diluted systemically in the recipient mice, only few cells will reach the tumors or other organs of interest and differentiate in situ into macrophages or dendritic cells (Figures 2 and 3). Hence, it is important to acquire many events by flow cytometry. After gating on the transferred CellTrace+ or GFP+ cells, the fate of their progeny can be determined using additional markers specific for monocytic myeloid-derived suppressor cells (Mo-MDSC), immature macrophages, M2-like tumor-associated macrophages (TAM), M1-like TAM or monocyte-derived dendritic cells (Laoui et al., 2016; Van Overmeire et al., 2016). For these type of experiments, two independent repeats containing each n ≥ 3 are generally accepted as many donor mice can be needed for acquiring enough cell in the receptor mice.


Figure 2. Adoptive transfer of CellTrace-labeled CD45.1+ monocytes in CD45.2 recipient mice. One million CellTrace-labeled CD45.1+ monocytes were adoptively transferred to 11-day old LLC tumor-bearing mice. Two days after CellTrace-labeled CD45.1+ monocytes transfer, mice were sacrificed and tumors were collected. Graphs show the percentage of GFP+Ly6Chi monocytes present in total tumors.


Figure 3. Adoptive transfer of GFP+ monocytes in CD45.2 recipient mice. One million GFP+ monocytes were adoptively transferred to 11-day old LLC tumor-bearing mice. Four hours after GFP+ monocyte transfer, mice were sacrificed, and tumors were collected. Graphs show the percentage of GFP+Ly6Chi monocytes present in total tumors.

Recipes

  1. 70% ethanol (for 100 ml)
    70 ml 99.9% ethanol (VWR Chemicals)
    30 ml demineralized water
  2. Complete medium
    Roswell Park Memorial Institute (RPMI)-1640
    10% (v/v) heat-inactivated fetal calf serum (FCS)
    300 μg•ml-1 L-glutamine
    100 U•ml-1 penicillin
    100 μg•ml-1 streptomycin
  3. Erythrocyte lysis buffer
    8.29 g•L-1 NH4Cl
    1 g•L-1 KHCO3
    37.2 mg•L-1 EDTA
    Bring at pH 7.2
  4. MACS buffer
    Hank’s buffered salt solution
    0.5% (v/v) heat-inactivated fetal calf serum
    2 mM EDTA 
  5. Sorting buffer
    Hank’s buffered salt solution
    0.5% (v/v) heat-inactivated FCS
    5 mM EDTA
  6. Violet tracer
    Resuspend CellTrace in 20 μl DMSO (provided)

Acknowledgments

The authors thank FWO-Vlaanderen, ‘Stichting tegen Kanker’ and ‘Komop tegen kanker’ for their support. This protocol was adapted from Laoui et al., (2016), Nat Comm, and Van Overmeire et al., (2016), Cancer Res. EVO and CA are supported by PhD grants from the Research Foundation Flanders (FWO). DL is supported by grants from Kom op tegen kanker and Vrije Universiteit Brussel. JVG is supported by grants from Kom op tegen kanker, FWO and Stichting tegen kanker.

Competing interests

The authors declare no competing financial interests.

Ethics

All procedures followed the guidelines of the Belgian Council for Laboratory Animal Science and were approved by the Ethical Committee for Animal Experiments of the Vrije Universiteit Brussel (licenses 11-220-3 and 15-220-2).

References

  1. Bolli, E., Movahedi, K., Laoui, D. and Van Ginderachter, J. A. (2017). Novel insights in the regulation and function of macrophages in the tumor microenvironment. Curr Opin Oncol 29(1): 55-61.
  2. Kiss, M., Van Gassen, S., Movahedi, K., Saeys, Y. and Laoui, D. (2018). Myeloid cell heterogeneity in cancer: not a single cell alike. Cell Immunol 330:188-201.
  3. Laoui, D., Keirsse, J., Morias, Y., Van Overmeire, E., Geeraerts, X., Elkrim, Y., Kiss, M., Bolli, E., Lahmar, Q., Sichien, D., Serneels, J., Scott, C. L., Boon, L., De Baetselier, P., Mazzone, M., Guilliams, M. and Van Ginderachter, J. A. (2016). The tumour microenvironment harbours ontogenically distinct dendritic cell populations with opposing effects on tumour immunity. Nat Commun 7: 13720.
  4. Laoui, D., Van Overmeire, E., Di Conza, G., Aldeni, C., Keirsse, J., Morias, Y., Movahedi, K., Houbracken, I., Schouppe, E., Elkrim, Y., Karroum, O., Jordan, B., Carmeliet, P., Gysemans, C., De Baetselier, P., Mazzone, M. and Van Ginderachte,r J. A. (2014). Tumor hypoxia does not drive differentiation of tumor-associated macrophages but rather fine-tunes the M2-like macrophage population. Cancer Res 74(1): 24-30.
  5. Movahedi, K., Laoui, D., Gysemans, C., Baeten, M., Stange, G., Van den Bossche, J., Mack, M., Pipeleers, D., In't Veld, P., De Baetselier, P. and Van Ginderachter, J. A. (2010). Different tumor microenvironments contain functionally distinct subsets of macrophages derived from Ly6C(high) monocytes. Cancer Res 70(14): 5728-5739.
  6. Van Overmeire, E., Stijlemans, B., Heymann, F., Keirsse, J., Morias, Y., Elkrim, Y., Brys, L., Abels, C., Lahmar, Q., Ergen, C., Vereecke, L., Tacke, F., De Baetselier, P., Van Ginderachter, J. A. and Laoui, D. (2016). M-CSF and GM-CSF receptor signaling differentially regulate monocyte maturation and macrophage polarization in the tumor microenvironment. Cancer Res 76(1): 35-42.

简介

炎症性Ly6C hi 单核细胞可在肿瘤微环境中产生不同的单核髓样细胞,如单核细胞源性抑制细胞(Mo-MDSC),未成熟巨噬细胞,M2样肿瘤相关巨噬细胞(TAMs) ),M1样TAM或单核细胞衍生的树突细胞(Mo-DC)。 该方案描述了评估肿瘤微环境中炎性Ly6C hi 单核细胞的命运和募集的方法。
【背景】肿瘤是异质微环境,其中在肿瘤细胞和浸润性炎性细胞(例如肿瘤相关巨噬细胞(TAM)和肿瘤相关树突细胞(TADC))之间发生复杂的相互作用。 临床研究强调了肿瘤浸润性单核骨髓细胞的相关性,显示其丰度与预后不良之间存在相关性(Bolli et al。,2007)。 TAM和TADC的起源一直存在争议,因为几个复杂程度导致相当大的TAM和TADC异质性(Movahedi et al。,2010; Laoui et al。 >,2014,Laoui et al。,2016; Van Overmeire et al。,2016; Kiss et al。,2018)。 在这里,我们描述了过继转移骨髓衍生的单核细胞的有价值的方法,允许评估它们在肿瘤中的募集和命运。

关键字:过继转移, 单核细胞, Pre-cDCs, 肿瘤相关巨噬细胞, 肿瘤相关树突状细胞, 细胞分选

材料和试剂

  1. 聚酯过滤器切成10 x 10厘米的正方形,螺纹直径70微米(Specturmlabs,目录号:146490)
  2. 10毫升注射器(Omnifix,目录号:473203)
  3. 1毫升注射器(Greiner,目录号:470203)
  4. 27 G针(BD Bioscience,目录号:300635)
  5. 25 G针(BD Biosciences,目录号:300400)
  6. 19 G针(BD Biosciences,目录号:301500)
  7. Falcon标准组织培养皿(Fisher Scientific,目录号:353003)
  8. BD Falcon 50 ml聚丙烯管(BD Biosciences,目录号:2070)
  9. BD Falcon 15 ml聚丙烯管(BD Biosciences,目录号:2096)
  10. BD Falcon 5 ml聚丙烯圆底管(BD Biosciences,目录号:352063)
  11. 70微米无菌尼龙纱布
  12. LS柱(Miltenyi,目录号:130-042-401)
  13. 幼稚小鼠:年龄最好在6到12周之间,应变可能因实验/项目而异(在本例中我们使用C57BL / 6小鼠)
  14. 乙醇绝对analaR Normapur ACS(VWR Chemicals,目录号:84857360)
  15. RPMI-1640培养基(RPMI)(Life Technologies,目录号:52400-041)
  16. 胎牛血清(FCS)(Life Technologies,Gibco,目录号:DE14-801F)
  17. L-谷氨酰胺(Life Technologies,目录号:25030-024)
  18. 青霉素 - 链霉素(Life Technologies,目录号:15140-130)
  19. 氯化铵(NH 4 Cl)(Merck KGaA,目录号:1011450500)
  20. 碳酸氢钾(KHCO 3 )(Merck KGaA,目录号:104852)
  21. EDTA(Duchefa Biochemie,目录号:E0511.1000)
  22. 汉克的缓冲盐溶液(HBSS)(Life Technologies,Gibco,目录号:14175129)
  23. 抗CD11b微珠(Miltenyi,目录号:130-049-601)
  24. 纯化的CD16 / CD32(FcBlock)(克隆2.4G2)(BD Biosciences,目录号:553142)
  25. PE-Cy7-缀合的抗-CD11b抗体(克隆M1 / 70)(BD Biosciences,目录号:552850) 
  26. AF647-缀合的抗-Ly6C抗体(克隆ER-MP20)(Serotec,目录号:MCA2389A647) 
  27. PerCP-Cy5.5-缀合的抗I-A / I-E(MHC-II)抗体(克隆M5 / 114.15.2)(BioLegend,目录号:107626) 
  28. FITC-缀合的抗-Ly6G抗体(克隆1A8)(BD Biosciences,目录号:551460) 
  29. APC-Cy7-缀合的抗CD45(克隆30-F11)(BioLegend,目录号:103116) 
  30. CellTrace Violet(Thermo Fisher Scientific,Molecular probes TM ,目录号:C34557)
  31. 台盼蓝(Life Technologies,Gibco,目录号:15250061)
  32. DMSO
  33. 70%乙醇(见食谱)
  34. 完全中等(见食谱)
  35. 红细胞裂解缓冲液(见食谱)
  36. MACS缓冲区(参见食谱)
  37. 排序缓冲区(参见食谱)
  38. 紫罗兰示踪剂(见食谱)

设备

  1. 无菌培养罩,PSM Optimale 18(ADS)
  2. 手术剪刀和镊子
  3. 37°C,5%CO 2 细胞培养箱(Binder,VWR)
  4. 移液器(吉尔森)
  5. 离心机5810 R(Eppendorf,型号:5810 R)
  6. 振动筛KS 260 Basic(IKA,型号:KS 260 basic)
  7. 显微镜Eclipse TS100(尼康,型号:Eclipse TS100)
  8. MidiMACS TM 分离器和MultiStand(Miltenyi,目录号:130-042-301)
  9. 多色FACS分拣机-FACS Aria II(BD Biosciences Aria流式细胞仪)

程序

  1. 骨髓单细胞悬液的制备
    1. 牺牲一只幼稚的老鼠,用注射器针将它的爪子钉在泡沫表面上来约束它。用70%乙醇消毒小鼠皮肤(见配方1)。从尾巴的基部到沿着小鼠腹部的颈部和爪子做一个平行的切口,不要刺穿腹膜。轻轻拉回皮肤并将其钉在泡沫表面以露出后肢(图1A)。
    2. 通过切入股骨 - 骨盆关节后面的骨盆,将后肢从皮肤和身体切开。保持股骨和胫骨整体。尝试用剪刀或手术刀尽可能多地去除骨头周围多余的组织(肌肉,纤维......)(图1B)。轻轻地做这个程序,以避免骨头断裂。 
    3. 通过向前和向后移动后爪轻轻拉动后肢(图1C)。 
    4. 通过将骨浸入70%乙醇中清洁骨,并将骨储存在冰上50ml Falcon管中的5ml完全培养基(参见配方2)中。
    5. 用第二个后肢重复此动作。
    6. 从股骨上分离胫骨并切除腓骨和髌骨并将骨头放入Falcon标准组织培养皿中(图1D,1E和1F)。
    7. 将27 G针头连接到10 ml注射器上,该注射器在股骨中含有完全培养基,并用10 ml完全培养基轻轻冲洗股骨(图1G和1H)。在冲洗之前,胫骨切开与爪子相邻的“白色”骨头,并将针头插入另一侧的骨头中。如果在冲洗骨骼时感觉到任何阻力,从末端切下一小部分骨头,并将针头重新插入剩余的骨头中。当所有红色骨髓都在盘子中时,冲洗完成,并且在骨骼中不再能看到红色组织。
    8. 通过(通过19G和1J)将含有骨髓的培养基通过(抽吸和压榨)两次使骨髓均质化(图1I和1J)。
    9. 将骨髓悬浮液通过70μm无菌尼龙纱布过滤到无菌50ml锥形管中,并用10ml完全培养基洗涤培养板和纱布。
    10. 将450 ml管在450 x g 下于4°C离心6分钟,弃去上清液(图1K)。
    11. 通过将沉淀重悬于5ml红细胞裂解缓冲液中(参见方法3)除去红细胞,并在室温下放置2分钟。 
    12. 通过加入15ml完全培养基中和,并通过70μm无菌尼龙纱布将悬浮液转移到新的50ml管中。 
    13. 将450ml管在450℃离心4℃,离心6分钟,弃去上清液(图1L)。
    14. 使用台盼蓝计数活细胞,并以10 8 细胞/ ml的浓度将细胞重悬于MACS缓冲液(参见配方4)中。从总骨髓中,通常10%至15%通常构成单核细胞(随年龄,小鼠品系和动物房类型略有不同),可以如下所述进行富集/纯化。


      图1.骨髓单细胞制备。 A.天真的小鼠。 B.带有爪子的后肢。 C.没有爪子的后肢。 D.没有爪子的清洁后肢。 E.从股骨上分离胫骨。 F. Femur(左)和tibia(右)。 G.冲洗骨头。 H.冲洗后立即骨髓。 I.骨髓的均质化。 J.均质化骨髓单细胞悬液。 K.红细胞裂解缓冲液前的细胞沉淀。 L.红细胞裂解缓冲液后的细胞沉淀。

  2. 从骨髓中纯化Ly6C 高单核细胞
    1. 每10 7 细胞加入5μl等分试样的抗CD11b磁性微珠,并在轨道振荡器上以4rpm在50rpm下孵育20分钟。
    2. 通过加入10ml MACS缓冲液洗涤,在4℃下以450×g离心6分钟并弃去上清液。
    3. 将LS柱置于与磁性MultiStand连接的MidiMACS TM 分离器中,并通过在顶部放置3ml MACS缓冲液进行洗涤。液体通过重力通过柱子。
    4. 将沉淀的细胞重悬于1ml MACS缓冲液中,并将标记的细胞悬浮液移液到LS分离柱的顶部。当细胞悬浮液通过柱子时,通过加入3×3ml MACS缓冲液洗涤柱子。
    5. 从分离器中取出LS柱,在柱顶部加入5 ml MACS缓冲液,然后通过用柱子上提供的柱塞紧紧冲洗柱子,用15 ml无菌管清洗磁性标记的细胞。
    6. 将CD11b + 细胞悬液与大鼠抗小鼠CD16 / CD32(每10 7 细胞10μg)在冰冷的水中孵育20分钟,以阻断Fc受体存在于细胞表面。
    7. 将细胞悬浮液与荧光标记的目标抗体(每10 7 细胞1μg)在冰冷的水中孵育另外20分钟,避免暴露在光线下。 
    8. 通过加入10ml MACS缓冲液洗涤,在4℃下以450×g离心6分钟并弃去上清液。
    9. 同时,预涂5毫升聚丙烯圆底管和15毫升含热灭活胎牛血清的试管,分别加入1毫升或2毫升热灭活的胎牛血清。用手轻轻摇动试管,使热灭活的胎牛血清覆盖试管的整个表面,并丢弃过量的热灭活的胎牛血清。这将防止细胞粘附在管上,从而增强细胞的恢复。
    10. 每10个 7 细胞将沉淀重悬于1ml分选缓冲液(参见配方5)中,并转移到预先涂有热灭活的胎牛血清的无菌5ml聚丙烯圆底管中。
    11. Ly6C 高单核细胞可以在BD FACS Aria上分类为CD45 pos CD11b pos Ly6G neg Ly6C 高 MHC-II neg 细胞(Laoui et al。,2016; Van Overmeire et al。,2016)。
    12. 将分选的单核细胞收集在预装有含3ml完全培养基的热灭活胎牛血清的15ml管中。

  3. 单体细胞标记体内跟踪
    1. 选项I:骨髓供体小鼠是野生型小鼠(Laoui 等人,,2016)。理想情况下,供体和受体的CD45等位基因不同(CD45.1与CD45.2)。
      1. 将含有分选的单核细胞的15ml管在450℃离心10分钟,并在4℃下离心10分钟,弃去上清液。
      2. 将细胞沉淀重悬于HBSS中,浓度为10 6 / ml,并加入1μlCellTrace(参见配方6)以染色1ml细胞(因此1:1,000稀释)。重要的是标记发生在无蛋白质培养基中。将细胞悬浮液在37°C孵育20分钟,避免暴露在光线下。
      3. 通过加入10ml HBSS洗涤,在4℃下以450×g离心6分钟并弃去上清液。
      4. 将细胞沉淀重悬于HBSS中,浓度为5×10 6 / ml,并保持在冰上直至进入动物设施。
      5. 使用25 G针头和1 ml注射器(视频1)在尾静脉中静脉注射200μl受体CD45.2小鼠。(视频1)。


        视频1.静脉尾静脉注射。 (该视频是根据比利时实验动物科学委员会的指导在布鲁塞尔自由大学制作的,并获得了布鲁塞尔自由大学动物实验伦理委员会的批准(许可证15-220-2)。

      6. 单核细胞的后代可以从接种后一天到10天(理想地48至72小时)进行追踪。可以通过流式细胞术在太平洋蓝色通道(405nm)中追踪单核细胞衍生的细胞。此外,CD45.1和CD45.2可用作互补染色。

    2. 方案二:骨髓供体小鼠是泛素-GFP小鼠(Van Overmeire et al。,2016)。
      1. 将含有分选的单核细胞的15ml管在450℃离心10分钟,并在4℃下离心10分钟,弃去上清液。
      2. 将细胞沉淀重悬于HBSS中,浓度为5×10 6 / ml,并保持在冰上直至进入动物设施。
      3. 使用25G针头和1ml注射器在尾静脉中静脉内注射200μl细胞的受体CD45.2小鼠(视频1)。
      4. 单核细胞的后代可以从接种后一天到10天(理想地48至72小时)进行追踪。可以通过流式细胞术在FITC通道中追踪单核细胞衍生的细胞。

数据分析

流式细胞术通常用于显现受体小鼠肿瘤中转移的单核细胞。当转移的单核细胞在受体小鼠中全身稀释时,只有少数细胞会到达肿瘤或其他感兴趣的器官并原位分化成巨噬细胞或树突细胞(图2和3)。因此,通过流式细胞术获得许多事件是很重要的。在转移的CellTrace + 或GFP + 细胞上进行门控后,可以使用特异于单核细胞髓源抑制细胞(Mo-MDSC)的其他标记来确定其后代的命运。 ,未成熟巨噬细胞,M2样肿瘤相关巨噬细胞(TAM),M1样TAM或单核细胞衍生的树突状细胞(Laoui et al。,2016; Van Overmeire et al。,2016)。对于这些类型的实验,通常接受两个独立的重复,其中每个n≥3,因为在受体小鼠中获得足够的细胞可能需要许多供体小鼠。


图2.在CD45.2受体小鼠中过继转移CellTrace标记的CD45.1 + 单核细胞。 将100万个CellTrace标记的CD45.1 + 单核细胞过继转移至11日龄的LLC荷瘤小鼠。在CellTrace标记的CD45.1 + 单核细胞转移两天后,处死小鼠并收集肿瘤。图表显示总肿瘤中存在的GFP + Ly6C hi 单核细胞的百分比。


图3. CD45.2受体小鼠中GFP + 单核细胞的过继转移。将100万GFP + 单核细胞过继转移至11日龄LLC荷瘤小鼠。 GFP + 单核细胞转移4小时后,处死小鼠,收集肿瘤。图表显示总肿瘤中存在的GFP + Ly6C hi 单核细胞的百分比。

食谱

  1. 70%乙醇(100毫升)
    70毫升99.9%乙醇(VWR Chemicals)
    30毫升软化水
  2. 完整媒体
    罗斯威尔公园纪念研究所(RPMI)-1640
    10%(v / v)热灭活的胎牛血清(FCS)
    300μg•ml -1 L-谷氨酰胺
    100 U•ml -1 青霉素
    100μg•ml -1 链霉素
  3. 红细胞裂解缓冲液
    8.29 g•L -1 NH 4 Cl
    1 g•L -1 KHCO 3
    37.2 mg•L -1 EDTA
    在pH 7.2下
  4. MACS缓冲区
    汉克的缓冲盐溶液
    0.5%(v / v)热灭活的胎牛血清
    2 mM EDTA 
  5. 排序缓冲区
    汉克的缓冲盐溶液
    0.5%(v / v)热灭活FCS
    5mM EDTA
  6. 紫罗兰追踪器
    将CellTrace重悬于20μlDMSO中(提供)

致谢

作者感谢FWO-Vlaanderen,'Stichting tegen Kanker'和'Komop tegen kanker'的支持。该协议改编自Laoui et al。,(2016), Nat Comm 和Van Overmeire et al。,(2016), Cancer Res 。 EVO和CA得到法兰克福研究基金会(FWO)的博士学位资助。 DL得到了Kom op tegen kanker和Vrije Universiteit Brussel的资助。 JVG得到了Kom op tegen kanker,FWO和Stichting tegen kanker的资助。

利益争夺

作者声明没有竞争性的经济利益。

伦理

所有程序均遵循比利时实验动物科学理事会的指导原则,并获得布鲁塞尔自由大学动物实验伦理委员会的批准(许可证11-220-3和15-220-2)。

参考

  1. Bolli,E.,Movahedi,K.,Laoui,D。和Van Ginderachter,J.A。(2017)。 关于肿瘤微环境中巨噬细胞调节和功能的新见解。 Curr Opin Oncol 29(1):55-61。
  2. Kiss,M.,Van Gassen,S.,Movahedi,K.,Saeys,Y。和Laoui,D。(2018)。 癌症中的髓样细胞异质性:不是单个细胞。 细胞免疫 330:188-201。
  3. Laoui,D.,Keirsse,J.,Morias,Y.,Van Overmeire,E.,Geeraerts,X.,Elkrim,Y.,Kiss,M.,Bolli,E.,Lahmar,Q.,Sichien,D。 ,Serneels,J.,Scott,CL,Boon,L.,De Baetselier,P.,Mazzone,M.,Guilliams,M。和Van Ginderachter,JA(2016)。 肿瘤微环境含有致癌性不同的树突状细胞群,对肿瘤免疫有相反的作用。 em> Nat Commun 7:13720。
  4. Laoui,D.,Van Overmeire,E.,Di Conza,G.,Aldeni,C.,Keirsse,J.,Morias,Y.,Movahedi,K。,Houbracken,I.,Schouppe,E.,Elkrim,Y ,Karroum,O.,Jordan,B.,Carmeliet,P.,Gysemans,C.,De Baetselier,P.,Mazzone,M。和Van Ginderachte,r JA(2014)。 肿瘤缺氧不会驱动肿瘤相关巨噬细胞的分化,而是微调M2样巨噬细胞人口。 癌症研究 74(1):24-30。
  5. Movahedi,K.,Laoui,D.,Gysemans,C.,Baeten,M.,Stange,G.,Van den Bossche,J.,Mack,M.,Pipeleers,D.,In't Veld,P。, De Baetselier,P。和Van Ginderachter,JA(2010)。 不同的肿瘤微环境包含源自Ly6C(高)单核细胞的功能不同的巨噬细胞亚群。 Cancer Res 70(14):5728-5739。
  6. Van Overmeire,E.,Stijlemans,B.,Heymann,F.,Keirsse,J.,Morias,Y.,Elkrim,Y.,Brys,L.,Abels,C.,Lahmar,Q.,Ergen,C。 ,Vereecke,L.,Tacke,F.,De Baetselier,P.,Van Ginderachter,JA and Laoui,D。(2016)。 M-CSF和GM-CSF受体信号通路差异调节肿瘤微环境中单核细胞成熟和巨噬细胞极化。 Cancer Res 76(1):35-42。
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引用:Laoui, D., Overmeire, E. V., Abels, C., Keirsse, J. and Ginderachter, J. A. V. (2019). Adoptive Transfer of Monocytes Sorted from Bone Marrow. Bio-protocol 9(1): e3134. DOI: 10.21769/BioProtoc.3134.
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