搜索

LT-HSC Methylcellulose Assay
长期造血干细胞(LT-HSC)的甲基纤维素试验   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Hematopoietic differentiation is a highly complex process originating from an extraordinary population of cells called long-term repopulating hematopoietic stem cells (LT-HSCs). The unique feature of all stem cells, including HSCs, is their exceptional ability to divide asymmetrically giving rise to two different kinds of offspring. One daughter cell becomes an LT-HSC itself (self-renews) to maintain the LT-HSC pool, whereas the second daughter cell pursues a differentiation fate to ultimately give rise to terminally differentiated mature blood cells (Orkin and Zon, 2008). Quantification of phenotypic LT-HSCs can be performed by multi-color flow cytometry and the gold standard for assessment of LT-HSC self-renewal and function is competitive bone marrow transplantation (Miller et al., 2008). Although these methods are irreplaceable to determine LT-HSC abundance and functionality, they have their disadvantages and limitations. For example, competitive bone marrow transplantation is typically monitored as a function of peripheral blood donor contribution over 12-16 weeks. While reduced peripheral blood donor contribution by itself signifies impairment in the stem/progenitor cells compartment, it cannot unambiguously discriminate between reduced LT-HSC self-renewal, impaired LT-HSC differentiation or compromised progenitor cell differentiation. Here we describe an LT-HSCs methylcellulose colony-forming assay, as a fast complementary in vitro method to directly assess LT-HSC differentiation capacity. As described in Kerenyi et al. (2013), this technique acts as a powerful tool to differentiate between LT-HSC or progenitor cell differentiation defects.

Keywords: HSC(HSC), Stem Cell(干细胞), Colony Assay(菌落检测), Methylcellulose(甲基纤维素)

Materials and Reagents

  1. Mice (C57BL/6, 12-16 weeks)
  2. Bone marrow cells from experimental mice
  3. 7-amino-actinomycin D (7AAD) (BD, catalog number: 559925 )
  4. CD150 Alexa488 antibody (BioLegend, catalog number: 115916 )
  5. CD48 APC antibody (eBiosciences, catalog number: 17048182 )
  6. cKit APC-efluor 780 antibody (eBiosciences, catalog number: 47117182 )
  7. DynaBeads Biotin Binder (Life Technologies, InvitrogenTM, catalog number: 11047 )
  8. EDTA (0.5 M, pH 8.0) (Boston BioProducts, catalog number: BM-150 )
  9. Fetal Bovine Serum (FBS) (Gemini Bio-Products, catalog number: 100106 )
  10. Ficoll-Paque Premium (General Electric Company, catalog number: 17544202 )
  11. Iscove’s Modified Dulbecco’s Medium (IMDM) (Gibco®, catalog number: 12440053 )
  12. L-Glutamine (Gibco®, catalog number: 25030081 )
  13. MethoCult GF M 3434 methylcellulose-based medium (STEMCELL Technologies, catalog number: 3434)
  14. Mouse Hematopoietic Lineage Biotin Panel (eBiosciences, catalog number: 88777475 )
  15. Phosphate buffered saline (PBS) without Ca2+ and Mg2+ (Mediatech, Cellgro®, catalog number: 21031CM )
  16. Penicillin/Streptomycin Solution (Gibco®, catalog number: 15140122 )
  17. Purified Anti CD16/CD32 antibody (FC Block) (BD, catalog number: 553142 )
  18. Recombinant mouse interferon (IFN) alpha A (R&D Systems, catalog number: 121001 )
  19. Sca-1 PE-Cy7 antibody (eBiosciences, catalog number: 25598182 )
  20. Streptavidin PerCP (BD, catalog number: 554064 )
  21. Staining Medium for FACS (see Recipes)
  22. IMDM for methylcellulose colony assays (see Recipes)

Equipment

  1. Mortar and pestle
  2. Easy Grip 35 mm petri dish (BD Biosciences, Falcon®, catalog number: 351008 )
  3. 6 well plate
  4. 1.5 ml Eppendorf Protein LoBind Tube (Eppendorf, catalog number: 022431081 )
  5. 1.5 ml Eppendorf Safe-Lock Tubes (Eppendorf, catalog number: 0030120086 )
  6. 2.0 ml Eppendorf Safe-Lock Tubes (Eppendorf, catalog number: 0030120094 )
  7. 40 µM BD Cell stainer (BD Biosciences, Falcon®, catalog number: 352340 )
  8. 15 ml BD Falcon Conical (BD Biosciences, Falcon®, catalog number: 352095 )
  9. 50 ml BD Falcon Conical (BD Biosciences, Falcon®, catalog number: 1443222 )
  10. 10 cm BD Falcon Tissue culture dish (BD Biosciences, Falcon®, catalog number: 353003 )
  11. 5 ml BD Polystyrene Tubes (FACS tubes) (BD Biosciences, Falcon®, catalog number: 352054 )
  12. BD PrecisionGlide Needle (16 gauge) (BD Biosciences, Falcon®, catalog number: 305198 )
  13. 3 ml BD Syringe Luer-Lok Tip (BD Biosciences, Falcon®, catalog number: 309657 )
  14. BD FACS Aria I (BD Biosciences, special order product)
  15. Inverted Light Microscope (OLYMPUS, catalog number: CKX31 )
  16. DynaMag-2 Magnet (Life Technologies, InvitrogenTM, catalog number: 12321D )

Procedure

Day 0

  1. Thaw sufficient numbers of 3 ml MethoCult GF M3434 aliquots overnight (one 3 ml aliquot per mouse) at 4 °C.

Day 1

  1. Euthanize mice according to the method of choice, e.g., cervical dislocation, CO2.
  2. Disinfect mice with 70% ethanol. Open skin on top of the femur medially (on the interior side of hindlegs). Using small scissors and forceps, dissect out ilii (hip bones), femora and tibiae from mice and place them in one well of a 6 well plate containing 2 ml ice-cold PBS/2% FBS 2 mM EDTA (= staining medium).
  3. Isolate bone marrow (BM) using autoclaved mortar and pestle: Put femora, tibiae, iliii of one mouse into mortar and add 5 ml staining medium and grind until bones are not red anymore. Filter cell suspension into a 50 ml conical tube through a 40 µM nylon mesh and use the plunger of a 5 ml syringe to push cells trough the mesh which are held back by bone marrow fat. Repeat at least 2 more times.
  4. Fill 50 ml conical tube up to 40 ml with staining medium and spin down at 300 x g at 4 °C.
  5. Resuspend BM cells in 5 ml staining medium.
  6. Fill 5 ml Ficoll at room temperature (!) into a 15 ml conical tube.
  7. Slowly overlay 5 ml of the bone marrow cell suspension onto the Ficoll.
  8. Spin down cells for 15 min at 400 x g at room temperature, with the brake off (or low). This will get rid of the bulk of erythroid and myeloid cells.
  9. Using a pasteur pipette transfer the interface, containing mononuclear cells, into another 15 ml tube.
    Note: The interface may not appear if Ficoll is not at room temperature or if the centrifuge chamber is chilled.
  10. Wash the bone marrow mononuclear cells with 15 ml staining medium.
  11. Spin down cells for 7 min at 300 x g at 4 °C and resuspend again in 10 ml staining medium. At this point a small aliquot can be drawn to enumerate the cell number.
  12. Spin down cells for 7 min at 300 x g at 4 °C. Aspirate supernatant completely. Resuspend pellet in 50 µl staining buffer.
  13. Add 1 µl of purified anti CD16/CD32 antibody (FC-Block) to keep FC-receptors from capturing antibodies, which would result in false positive staining. Incubate for 15 min in the fridge.
  14. Add 50 µl of biotinylated lineage marker antibody cocktail (Mouse Hematopoietic Lineage Flow Panel):
    1x lineage marker cocktail:
    Biotinylated anti-Ter-119 (2 µl/1 x 107 cells)
    Biotinylated anti-Gr-1 (2 µl/1 x 107 cells)
    Biotinylated anti-Mac-1 (2 µl/1 x 107 cells)
    Biotinylated anti-B220 (2 µl/1 x 107 cells)
    Biotinylated anti-CD3 (2 µl/1 x 107 cells)
    Up to 50 µl with staining buffer
  15. Incubate cells in fridge for 30 min.
  16. Wash cells once with 5 ml of staining buffer and spin down for 5 min at 300 x g at 4 °C.
  17. Resuspend cells in 1.5 ml staining medium (in 2 ml Eppendorf tube).
  18. Preparation for DynaBeads Biotin Binder for lineage depletion:
    a.  Resuspend the Dynabeads in the vial.
    b.  Transfer the desired volume of Dynabeads to a 1.5 ml tube.
    c.  Add the same volume of staining buffer.
    d.  Place the tube in a magnet for 3 min and discard the supernatant.
    e.  Repeat steps c and d twice.
    f.   Remove tubes from magnet and resuspend washed Dynabeads in the same volume of staining buffer as the starting volume of beads was.
  19. Add ~300 µl DynaBeads Biotin Binder (or 4 Dynabeads per target cell) per mouse and incubate cells rotating at 2-8 °C for 30 min.
  20. Put tubes containing the cells in magnet for 3 min. Transfer supernatant into FACS tube.
  21. Wash cells once with 10 ml of staining buffer and resuspend in 50 µl staining buffer.
  22. Add 50 µl of HSC staining cocktail and stain 30 min at 4 °C. Flick sample every 10 min to mix.
    1x HSC staining cocktail:
    Streptavidin PerCP (0.125 µg/1 x 107 cells)
    CD150-Alexa488 (0.5 µg/1 x 107 cells)
    CD48-APC (0.125 µg/1 x 107 cells)
    cKit-APC-eFluor780 (0.125 µg/1 x 107 cells)
    Sca1-PE-Cy7 (0.125 µg/1 x 107 cells)
    Biotinylated anti-Ter-119 Ab (2 µl/1 x 107 cells)
    Biotinylated anti-Gr-1 Ab (2 µl/1 x 107 cells)
    Biotinylated anti-Mac-1 Ab (2 µl/1 x 107 cells)
    Biotinylated anti-B220 Ab (2 µl/1 x107 cells)
    Biotinylated anti-CD3 Ab (2 µl/1 x 107 cells)
    Up to 50 µl with staining buffer
  23. Wash cells once with 3 ml staining buffer, spin down for 5 min at 300 x g at 4 °C and resuspend in 300 µl staining buffer.
  24. Add 3 µl 7-AAD and incubate 10 min at room temperature.
    Note: The LT-HSC isolation part of this protocol has been adapted and modified from Ema et al. (2006).
  25. Sort cells on a BD FACS ARIA I (or comparable Cell Sorter). To achieve over 95% purity of LT-HSCs, perform 2 rounds of sorting! See Figure 1 for gating strategy.
    1. 1st sort: Sort at least 5000 LT-HSCs (i.e. lineage negative Sca-1+ c-Kit+ CD150+CD48- cells) into 250 µl of IMDM 2% FCS + 1% P/S in a low protein binding 1.5 ml Eppendorf tube. Using a low protein binding tube at this step will maximize your LT-HSC recovery.
    2. 2nd sort: Sort exactly 1000 LT-HSCs into 500 µl of IMDM 2% FCS + 1% P/S in a low protein binding 1.5 ml Eppendorf tube. You will now have 200 LT-HSCs/100 µl.
    Optional: If using Mx1Cre BM you can either delete your floxed allele in vivo with pIpC (not described here) or in vitro using 1,000 U/ml recombinant mouse Interferon alpha (IFNα). In vitro deletion may be desirable in cases in which knock out of your gene of interest results in altered cell surface immunophenotypes.
    Note: The specific activity of IFNα varies from Lot to Lot. Final volume of IFNα to obtain 3,000 U/3 ml Methocult aliquots will have to be adjusted for every lot.


    Figure 1. Gating strategy for the isolation of CD150+ LT-HSCs. A. Bone marrow cells were stained with indicated antibodies and sorted by flow cytometry to isolate CD150+ LT-HSCs.

  26. A total of 300 µl volume can be added to 3 ml Methocult M3434 aliquot:
    60 µl HSCs (from 200 LT-HSCs/100 µl) = about 40 LT-HSCs/ml Methocult M3434
    30 µl Pen/Strep
    Optional: x µl IFNα (Lot dependent–but a total of 3,000 U)
    Up to 300 µl with IMDM 2% FCS 1% Pen/Strep 1% L-Glut
  27. Mix well by vortex, and let methylcellulose settle at least for 5 min to allow air bubbles to dissipate.
  28. Plate 2 x 1.1 ml of Methocult M3434 including your LT-HSCs per 35 mm petri dish using a 3 CC syringe with a 16 gauge needle. You now have technical duplicates of your colony assay.
    Note: You have to use a petri dish not a tissue culture dish!
  29. Place the two dishes into a 100 mm petri dish. Add a third, uncovered 35 mm dish containing 3 ml of sterile water. Replace lid of 100 mm petri dish.
  30. Transfer the duplicate samples and your petri dish filled with water into a 10 cm tissue culture grade dish and put the lid on it. This installation will keep your methylcellulose from drying out. Transfer to tissue culture incubator and incubate for 10-12 days at 37 °C.

Day 10-12

  1. Using an inverted light microscope, count and score the number of colonies according to the instructions found in Miller et al. (2008). You should largely obtain large multipotential CFU-GEMM (Colony Forming Unit–granulocyte, erythrocyte, monocyte, megakaryocyte) colonies (Figure 2A). When seeding 40 wild type LT-HSCs we typically observe ~20 CFU-GEMM colonies. If desired, cells can be harvested from methylcellulose and either subjected to cytospin analysis to determine cell morphologies (Figure 2B) or to flow cytometry, to obtain quantitative cell type distribution enumeration.


    Figure 2. Microphotographs of CFU-GEMM Colonies. A. Microphotograph if a single CFU-GEMM colony (scale bar = 500 µM). B. Microphotograph of a cytospun CFU-GEMM colony stained with hematoxylin and eosin (scale bar = 40 µM).

Recipes

  1. Staining Medium for FACS (for 500 ml)
    488 ml PBS (without Ca2+ or Mg2+)
    10 ml FBS (final concentration: 2% FBS)
    2 ml 0.5 M EDTA pH 8 (final concentration: 2 mM EDTA)
  2. IMDM for methylcellulose colony assays (for 500 ml)
    480 ml IMDM
    10 ml FBS (final concentration: 2% FBS)
    5 ml Pen/Strep (final concentration: 1% Pen/Strep)
    5 ml L-Glutamine (final concentration: 1% L-Glut)

Acknowledgments

This protocol was originally described in and adapted from Kerenyi et al. (2013). This work was supported by the NIH National Heart, Lung and Blood Institute, R01HL075735 and Austrian Science Fund (FWF), J 2948-B19. I would like to thank my mentor Stuart Orkin for giving me the opportunity to conceive, design and author this protocol.

References

  1. Ema, H., Morita, Y., Yamazaki, S., Matsubara, A., Seita, J., Tadokoro, Y., Kondo, H., Takano, H. and Nakauchi, H. (2006). Adult mouse hematopoietic stem cells: purification and single-cell assays. Nat Protoc 1(6): 2979-2987. 
  2. Kerenyi, M. A., Shao, Z., Hsu, Y. J., Guo, G., Luc, S., O'Brien, K., Fujiwara, Y., Peng, C., Nguyen, M. and Orkin, S. H. (2013). Histone demethylase Lsd1 represses hematopoietic stem and progenitor cell signatures during blood cell maturation. Elife 2: e00633. 
  3. Miller, C. L., Dykstra, B. and Eaves, C. J. (2008). Characterization of mouse hematopoietic stem and progenitor cells. Curr Protoc Immunol Chapter 22: Unit 22B 22.
  4. Orkin, S. H. and Zon, L. I. (2008). Hematopoiesis: an evolving paradigm for stem cell biology. Cell 132(4): 631-644. 

简介

造血分化是一种非常复杂的过程,源自称为长期重建造血干细胞(LT-HSCs)的非凡细胞群。所有干细胞(包括HSC)的独特之处在于其不对称的划分能够产生两种不同种类的后代。一个子细胞成为LT-HSC本身(自我更新)以维持LT-HSC库,而第二个子细胞追求分化命运,最终产生终末分化的成熟血细胞(Orkin和Zon,2008)。表型LT-HSC的定量可以通过多色流式细胞术进行,用于评估LT-HSC自我更新和功能的黄金标准是竞争性骨髓移植(Miller等,2008)。尽管这些方法对于确定LT-HSC丰度和功能是不可替代的,但它们具有其缺点和局限性。例如,竞争性骨髓移植通常被监测为12-16周内外周血献血者贡献的函数。虽然减少的外周血供体贡献本身表示干/祖细胞隔室中的损伤,但是它不能明确区分降低的LT-HSC自我更新,受损的LT-HSC分化或受损的祖细胞分化。这里我们描述一种LT-HSCs甲基纤维素集落形成测定法,作为直接评估LT-HSC分化能力的快速互补体外方法。如Kerenyi等人所述(2013),该技术是区分LT-HSC或祖细胞分化缺陷的有力工具。

关键字:HSC, 干细胞, 菌落检测, 甲基纤维素

材料和试剂

  1. 小鼠(C57BL/6,12-16周)
  2. 来自实验小鼠的骨髓细胞
  3. 7-氨基 - 放线菌素D(7AAD)(BD,目录号:559925)
  4. CD150 Alexa488抗体(BioLegend,目录号:115916)
  5. CD48 APC抗体(eBiosciences,目录号:17048182)
  6. cKit APC-efluor 780抗体(eBiosciences,目录号:47117182)
  7. DynaBeads生物素结合剂(Life Technologies,Invitrogen TM ,目录号:11047)
  8. EDTA(0.5M,pH8.0)(Boston BioProducts,目录号:BM-150)
  9. 胎牛血清(FBS)(Gemini Bio-Products,目录号:100106)
  10. Ficoll-Paque Premium(通用电气公司,目录号:17544202)
  11. Iscove's Modified Dulbecco's Medium(IMDM)(Gibco ,目录号:12440053)
  12. L-谷氨酰胺(Gibco ,目录号:25030081)
  13. MethoCult GF M3434甲基纤维素基培养基(STEMCELL Technologies,目录号:3434)
  14. 小鼠造血谱系生物素板(eBiosciences,目录号:88777475)
  15. 不含Ca 2+和Mg 2+的磷酸盐缓冲盐水(PBS)(Mediatech,Cellgro ,目录号:21031CM)
  16. 青霉素/链霉素溶液(Gibco ,目录号:15140122)
  17. 纯化的抗CD16/CD32抗体(FC Block)(BD,目录号:553142)
  18. 重组小鼠干扰素(IFN)αA(R& D Systems,目录号:121001)
  19. Sca-1PE-Cy7抗体(eBiosciences,目录号:25598182)
  20. 链霉亲和素PerCP(BD,目录号:554064)
  21. FACS的染色介质(参见配方)
  22. IMDM用于甲基纤维素菌落测定(参见配方)

设备

  1. 砂浆和杵
  2. Easy Grip 35mm培养皿(BD Biosciences,Falcon ,目录号:351008)
  3. 6孔板
  4. 1.5ml Eppendorf Protein LoBind Tube(Eppendorf,目录号:022431081)
  5. 1.5ml Eppendorf Safe-Lock Tubes(Eppendorf,目录号:0030120086)
  6. 2.0ml Eppendorf Safe-Lock Tube(Eppendorf,目录号:0030120094)
  7. 40μMBD细胞染色剂(BD Biosciences,Falcon ,目录号:352340)
  8. 15ml BD Falcon Conical(BD Biosciences,Falcon ,目录号:352095)
  9. 50ml BD Falcon Conical(BD Biosciences,Falcon ,目录号:1443222)
  10. 10cm BD Falcon组织培养皿(BD Biosciences,Falcon ,目录号:353003)
  11. 5ml BD聚苯乙烯管(FACS管)(BD Biosciences,Falcon ,目录号:352054)
  12. BD PrecisionGlide针(16号)(BD Biosciences,Falcon ,目录号:305198)
  13. 3ml BD注射器Luer-Lok Tip(BD Biosciences,Falcon ,目录号:309657)
  14. BD FACS Aria I(BD Biosciences,特殊订单产品)
  15. 倒置光显微镜(OLYMPUS,目录号:CKX31)
  16. DynaMag-2磁体(Life Technologies,Invitrogen TM ,目录号:12321D)

程序

第0天

  1. 在4℃下将足够数量的3ml MethoCult GF M3434等分试样溶解过夜(每只小鼠一个3ml等分试样)。

第1天

  1. 根据选择的方法,例如,颈部脱臼,CO 安乐死小鼠。
  2. 用70%乙醇消毒小鼠。 在股骨的顶部打开皮肤(在后腿的内侧)。 使用小剪刀和镊子,从小鼠中分离出ilii(髋骨),股骨和胫骨,并将其放置在含有2ml冰冷的PBS/2%FBS 2mM EDTA(=染色培养基)的6孔板的一个孔中。
  3. 使用高压灭菌的研钵和杵分离骨髓(BM):将一只小鼠的股骨,胫骨,iliii放入研钵中,加入5ml染色培养基并研磨直到骨不再是红色。 通过40μM尼龙网将细胞悬浮液过滤到50ml锥形管中,并使用5ml注射器的柱塞将细胞推过网状物,这些细胞被骨髓脂肪阻滞。 重复至少2次以上。
  4. 用染色介质填充至40ml的50ml锥形管,并在4℃下以300×g离心。
  5. 将BM细胞重悬在5ml染色培养基中
  6. 将5ml室温下的Ficoll(!)加入15ml锥形管中
  7. 将5ml骨髓细胞悬浮液缓慢地覆盖在Ficoll上
  8. 在室温下,在400×g下旋转电池15分钟,制动关闭(或低)。 这将摆脱大量的红细胞和骨髓细胞
  9. 使用巴斯德吸管将含有单核细胞的界面转移到另一个15ml管中。
    注意:如果Ficoll不在室温下或离心机室冷藏,界面可能不会出现。
  10. 用15ml染色培养基洗涤骨髓单核细胞
  11. 在4℃下以300×g离心细胞7分钟,并再次重悬于10ml染色培养基中。在这一点上,可以绘制小的等分来枚举单元格号
  12. 在4℃下以300×g离心细胞7分钟。完全吸出上清液。在50μl染色缓冲液中重悬沉淀
  13. 加入1μl纯化的抗CD16/CD32抗体(FC块),以防止FC受体捕获抗体,这将导致假阳性染色。在冰箱中孵育15分钟。
  14. 加入50μl生物素化的谱系标志物抗体混合物(小鼠造血谱系流面板):
    1x谱系标记鸡尾酒:
    生物素化的抗Ter-119(2μl/1×10 7个细胞)
    生物素化的抗Gr-1(2μl/1×10 7个细胞)
    生物素化的抗Mac-1(2μl/1×10 7个细胞)
    生物素化的抗B220(2μl/1×10 7个细胞)
    生物素化的抗CD3(2μl/1×10 7个细胞)
    用染色缓冲液高达50μl
  15. 孵育细胞在冰箱30分钟。
  16. 用5ml染色缓冲液洗涤细胞一次,并在4℃下以300×g离心5分钟。
  17. 将细胞重悬于1.5ml染色培养基(在2ml Eppendorf管中)
  18. DynaBeads生物素绑定物用于谱系衰竭的准备:
    a。  将Dynabeads重新悬挂在小瓶中。
    b。  将所需体积的Dynabeads转移到1.5 ml管 c。  加入相同体积的染色缓冲液。
    d。 将管在磁铁中放置3分钟,弃去上清液 e。  重复步骤c和d两次 f。   从磁体中取出管,并在与珠的起始体积相同体积的染色缓冲液中重悬悬洗涤的Dynabeads。
  19. 每个小鼠加入〜300μlDynaBeads生物素结合剂(或每个目标细胞4 Dynabeads),孵育细胞在2-8°C旋转30分钟。
  20. 将含有细胞的管在磁体中放置3分钟。 将上清转移到FACS管中
  21. 用10ml染色缓冲液洗涤细胞一次,并重悬于50μl染色缓冲液中
  22. 加入50μl的HSC染色鸡尾酒和染色30分钟,在4℃。 每10分钟轻拂样品混合。
    1x HSC染色混合物:
    链霉亲和素PerCP(0.125μg/1×10 7个细胞) CD150-Alexa488(0.5μg/1×10 7个细胞) CD48-APC(0.125μg/1×10 7个细胞) cKit-APC-eFluor780(0.125μg/1×10 7个细胞)
    Sca1-PE-Cy7(0.125μg/1×10 7个细胞) 生物素化的抗Ter-119 Ab(2μl/1×10 7个细胞)
    生物素化的抗Gr-1 Ab(2μl/1×10 7个细胞)
    生物素化的抗Mac-1 Ab(2μl/1×10 7个细胞)
    生物素化的抗B220 Ab(2μl/1×10 7个细胞)
    生物素化的抗CD3 Ab(2μl/1×10 7个细胞)
    用染色缓冲液高达50μl
  23. 用3ml染色缓冲液洗涤细胞一次,在4℃下以300×g离心5分钟,并重悬于300μl染色缓冲液中。
  24. 加入3微升7-AAD,在室温下孵育10分钟 注意:本协议的LT-HSC隔离部分已经适应和修改了Ema等人。 (2006)。
  25. 在BD FACS ARIA I(或类似的细胞分选仪)上分选细胞。为了实现超过95%的LT-HSC纯度,进行2轮分选!参见图1的门控策略。
    1. 1 st sort:至少排序5000个LT-HSC(即谱系阴性Sca-1 + c-Kit +
    2. 2 nd 分类:在低蛋白结合的1.5ml Eppendorf管中将1000个LT-HSC精确地分入500μl的IMDM 2%FCS + 1%P/S中。您现在将有200个LT-HSC/100μl。
    可选:如果使用Mx1Cre BM,您可以使用1,000 U/ml重组小鼠干扰素α(IFNα)通过pIpC(未在此处描述)或体外在体内删除floxed等位基因 )。在感兴趣的基因的敲除导致改变的细胞表面免疫表型的情况下,可能需要体外缺失。
    注意:IFNα的比活性因批次而异。最终体积的IFNα获得3,000U/3ml Methocult等分试样将必须对每一批进行调整。


    图1.用于分离CD150 + LT-HSC的门控策略 A.骨髓细胞染色并用流式细胞术分选以分离CD150 + sup/LT-HSC。

  26. 可将总共300μl体积加入到3ml Methocult M3434等分试样:
    中 60μlHSC(来自200个LT-HSC /100μl)=约40个LT-HSC/ml Methocult M3434
    30μlPen/Strep
    可选:xμlIFNα(批次依赖,但总共3,000 U)
    使用IMDM 2%FCS 1%Pen/Strep 1%L-Glut
    可达300μl
  27. 通过涡旋充分混合,让甲基纤维素沉降至少5分钟,以使气泡消散
  28. 板2×1.1ml Methocult M3434,包括您的LT-HSC/35mm培养皿,使用具有16号针的3cc注射器。 您现在有了您的菌落测定的技术副本。
    注意:您必须使用培养皿,而不是组织培养皿!
  29. 将两个菜放入100毫米培养皿。 添加第三,未覆盖35毫米的菜,含有3毫升无菌水。 更换100 mm培养皿的盖子。
  30. 将重复的样品和您的培养皿充满水转移到10厘米组织培养级菜,并把盖子上。 这个安装将保持你的甲基纤维素干燥。 转移到组织培养孵育器,并在37℃孵育10-12天

第10-12天

  1. 使用倒置光学显微镜,根据Miller等人(2008)中发现的说明对集落数进行计数和评分。您应该大量获得大型多能性CFU-GEMM(集落形成单位 - 粒细胞,红细胞,单核细胞,巨核细胞)集落(图2A)。当播种40野生型LT-HSC时,我们通常观察到〜20CFU-GEMM集落。如果需要,可以从甲基纤维素收获细胞,并且进行细胞离心涂片分析以确定细胞形态(图2B)或流式细胞术,以获得定量细胞类型分布计数。

    图2. CFU-GEMM集落的显微照片。 A.如果单个CFU-GEMM集落(比例尺=500μM)的显微照片。 B.用苏木精和曙红染色的细胞旋转CFU-GEMM集落的显微照片(比例尺=40μM)。

食谱

  1. 用于FACS的染色培养基(500ml)
    488ml PBS(无Ca 2+ +/Mg 2+或Mg 2+ 2 + )。
    10ml FBS(终浓度:2%FBS) 2ml 0.5M EDTA pH8(终浓度:2mM EDTA)
  2. IMDM用于甲基纤维素菌落测定(500ml) 480 ml IMDM
    10ml FBS(终浓度:2%FBS) 5ml Pen/Strep(终浓度:1%Pen/Strep) 5ml L-谷氨酰胺(终浓度:1%L-Glut)

致谢

该协议最初在Kerenyi等人(2013)中描述和改编。 这项工作由NIH国家心脏,肺和血液研究所,R01HL075735和奥地利科学基金(FWF),J 2948-B19支持。 我要感谢我的导师Stuart Orkin给我机会来设想,设计和创作这个协议。

参考文献

  1. Ema,H.,Morita,Y.,Yamazaki,S.,Matsubara,A.,Seita,J.,Tadokoro,Y.,Kondo,H.,Takano,H。和Nakauchi, 成人小鼠造血干细胞:纯化和单细胞测定。 Nat Protoc 1(6):2979-2987。 
  2. Kerenyi,MA,Shao,Z.,Hsu,YJ,Guo,G.,Luc,S.,O'Brien,K.,Fujiwara,Y.,Peng,C.,Nguyen,M.and Orkin, )。 组蛋白去甲基化酶Lsd1抑制血细胞成熟过程中的造血干细胞和祖细胞特征。 Elife 2:e00633。
  3. Miller,C.L.,Dykstra,B。和Eaves,C.J。(2008)。 小鼠造血干细胞和祖细胞的表征。 Curr Protoc Immunol Chapter 22:Unit 22B 22.
  4. Orkin,S.H。和Zon,L.I。(2008)。 血细胞生成:干细胞生物学的演进模式。 132(4):631-644。
  • 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 Kerenyi. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Kerenyi, M. A. (2014). LT-HSC Methylcellulose Assay. Bio-protocol 4(5): e1067. DOI: 10.21769/BioProtoc.1067.
  2. Kerenyi, M. A., Shao, Z., Hsu, Y. J., Guo, G., Luc, S., O'Brien, K., Fujiwara, Y., Peng, C., Nguyen, M. and Orkin, S. H. (2013). Histone demethylase Lsd1 represses hematopoietic stem and progenitor cell signatures during blood cell maturation. Elife 2: e00633.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。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或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。