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Adhesion Assay for Murine Bone Marrow Hematopoietic Stem Cells
鼠骨髓造血干细胞粘附测定   

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Abstract

Hematopoietic stem cells (HSCs) are defined by their functional abilities to self-renew and to give rise to all mature blood and immune cell types throughout life. Most HSCs are retained in a non-motile quiescent state within a specialized protective microenvironment in the bone marrow (BM) termed the niche. HSCs are typically distinguished from other adult stem cells by their motility capacity. Movement of HSCs across the physical barrier of the marrow extracellular matrix and blood vessel endothelial cells is facilitated by suppression of adhesion interactions, which are essential to preserve the stem cells retained within their BM niches. Importantly, homing of HSCs to the BM following clinical transplantation is a crucial first step for the repopulation of ablated BM as in the case of curative treatment strategies for hematologic malignancies. The homing process ends with selective access and anchorage of HSCs to their specialized niches within the BM. Adhesion molecules are targets to either enhance homing in cases of stem cell transplantation or reduce BM retention to harvest mobilized HSCs from the blood of matched donors. A major adhesion protein which is functionally expressed on HSCs and is involved in their homing and retention is the integrin alpha4beta1 (Very late antigen-4; VLA4). In this protocol we introduce an adhesion assay optimized for VLA4 expressing murine bone marrow stem cells. This assay quantifies adherent HSCs by flow cytometry with HSC enriching cell surface markers subsequent to the isolation of VLA4 expressing adherent cells.

Keywords: Very late antigen 4(极晚期抗原4), Integrin alpha4beta1(整合素α4β1), Adhesion-assay(粘附测定), Endothelial protein C receptor (EPCR)(内皮蛋白C受体(EPCR)), Long-term repopulating hematopoietic stem cells (LT-HSC)(长期再生造血干细胞(LT-HSC)), Flow cytometry(流式细胞术)

Background

HSCs are mostly retained in the BM and are regulated by adhesive interactions with their microenvironment, the niche. In this way HSCs are kept in a non-motile quiescent state which protects them from DNA damaging agents (Boulais and Frenette, 2015; Mendelson and Frenette, 2014; Miyamoto et al., 2011; Morrison and Scadden, 2014). The defining properties of HSCs are their functional ability to durably repopulate the irradiated BM of transplanted recipients, which requires their homing, self-renewal and developmental potential (Gur-Cohen et al., 2016). Since adhesion gives rise to activation of intracellular signaling pathways, the type of interaction can mirror the developmental state and behavior of the cells (Sugiyama et al., 2006). Adhesion assays are methods to distinguish between adhesive and non-adhesive cells. In this protocol we introduce a cell adhesion assay under static conditions that separates VLA4 expressing adhesive cells from non-adhesive cells, which are quantified by FACS analysis.
   In mouse, hematopoietic stem and progenitor cells (HSPCs) are enriched in a population that lacks lineage markers (Lin; CD8a, CD4, GR1, B220, TER-119, CD11b), and expresses c-Kit (K) and Sca-1 (S). Hence, these cells are also called Lin- Sca-1+ c-Kit+ (LSK) cells (Adolfsson et al., 2001; Okada et al., 1991; Spangrude et al., 1988). EPCR (endothelial protein C receptor) has been identified as a stem cell marker also in various other tissues (Balazs et al., 2006; Iwasaki et al., 2010; Kent et al., 2009; Ramalho-Santos et al., 2002; Wang and Gerdes, 2015).
   Adhesion molecules play a major role in the retention and egress of these HSCs in the BM and to the blood circulation. VLA4 is a receptor for both fibronectin and VCAM-1 and is expressed by most leukocytes, as well as by some non-hematopoietic cells (Hemler et al., 1990), while its expression is higher on murine BM EPCR+ LT-HSCs as compared to EPCR negative progenitor cells and circulating LT-HSC (Gur-Cohen et al., 2015). It has long been proposed that VLA4 expression by LT-HSCs might be important for binding and detachment of stem cells within the human BM microenvironment. Inhibition of VLA4 or VCAM-1 binding by neutralizing antibodies causes mobilization of HSPCs from the BM to the blood circulation of mice and primates (Craddock et al., 1997; Papayannopoulou et al., 1995) which is consistent with the notion that VLA4 is crucial for CXCL12/CXCR4-mediated LT-HSC quiescent retention in the BM (Papayannopoulou et al., 1995; Papayannopoulou and Scadden, 2008). In addition to HSPC BM retention, VLA4 is also essential for murine HSPC BM homing (Papayannopoulou and Craddock, 1997). VLA4 possesses different conformations that correlate with its affinity states (Alon et al., 1995; Chen et al., 1999; Feigelson et al., 2001) which are influenced by divalent cations and inside-out signaling (Chigaev et al., 2003; Chigaev et al., 2011). The majority of VLA4 affinity inside- out signaling is mediated by G-protein coupled receptors (Laudanna et al., 2002; Chigaev et al., 2008; Arnaout et al., 2007). Furthermore, elevation of intracellular nitric oxide (NO) was shown to cause cGMP-mediated inhibition of VLA4 affinity (Chigaev et al., 2011). We have previously shown two different pathways, the aPC-EPCR-PAR1 and the thrombin-PAR1 axis, which regulate the NO level up and down, respectively. Thereby, these pathways influence a number of intracellular molecules including Cdc42, CXCR4 and VLA4 leading to retention or mobilization of HSPCs (Gur-Cohen et al., 2015). As described by Gur-Cohen et al. (2015), we herein propose the VLA4 mediated adhesion assay for EPCR+ stem cells as a powerful tool to predict LT-HSC retention potential to their bone marrow niches.

Materials and Reagents

  1. Tissue culture 6-well plates (Corning, Costar®, catalog number: 3516 )
  2. NuncTM 8.8 cm2 Petri dish (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 153066 )
  3. 1 ml slip tip Sub-Q syringe with disposable 26 G x 5/8 inch needle (BD, catalog number: 309597 )
  4. Autoclaved pipet tips :
    1. 200-1,000 µl blue, gilson tip (Greiner Bio One, catalog number: 740290 )
    2. 200 µl universal tip (Neptune Scientific, catalog number: 2100.N )
    3. 0.5-10 µl pipet tips (Corning, Axygen®, catalog number: T-300 )
  5. FACS tubes (Corning, Falcon®, model: 352054 )
  6. Sterilized 40 µm pore-sized nylon mesh (Sinun Tech, catalog number: Plymer Screends ) (see Recipes)
  7. Mice
  8. Fibronectin (Sigma-Aldrich, catalog number: F0895 )
  9. Human CXCL12 (human SDF-1 alpha) (reprokineTM, catalog number: RKP48061 ) or (PeproTech, Rocky Hill, NJ, USA)
  10. BSA (Sigma-Aldrich, catalog number: A9647 )
  11. LymphoprepTM Ficoll (STEMCELL Technologies, catalog number: 0 7861 )
  12. Antibodies:
    1. EPCR PE (Affymetrix, eBioscience, catalog number: 12-2012-82 ) or EPCR PerCP-eFluor 710 (Affymetrix, eBioscience, catalog number: 46-2012-80 ) or EPCR Biotin (Affymetrix, eBioscience, catalog number: 13-2012-82 ) combined with streptavidin-PE (BioLegend, catalog number: 405203 )
    2. Sca-1 PE (BioLegend, catalog number: 108108 ) or Sca-1 PE/Cy7 (BioLegend, catalog number: 108114
    3. c-Kit APC (BioLegend, catalog number: 105812 )
    4. Lineage antibodies
      Note: This is a set of antibodies that target the antigens i-vi listed below which are cell lineage markers. In mice these markers do not occur on stem and progenitor cells.
      1. CD8a FITC (BioLegend, catalog number: 100706 )
      2. CD4 FITC (BioLegend, catalog number: 100406 )
      3. GR1 FITC (BioLegend, catalog number: 108406 )
      4. B220 FITC (BioLegend, catalog number: 103206 )
      5. TER-119 FITC (BioLegend, catalog number: 116206 )
      6. CD11b FITC (BioLegend, catalog number: 101206 )
      7. Or Lineage Cocktail-Biotin (Miltenyi Biotec, catalog number: 130-092-613 ) combined with streptavidin-FITC (BioLegend; catalog number: 405202 )
        Note: The combination of fluorophores used for the experiment needs to be adjusted according to the experimental demands.
  13. 1x Dulbecco’s phosphate buffered saline without calcium and magnesium (PBS-/-) (generated from 10x PBS, see Recipes) (Biological Industries, catalog number: 02-023-5A )
  14. Roswell park memorial institute (RPMI) 1640 medium ([+] 300 mg/L L-glutamine, [+] 25 mM HEPES) (Thermo Fisher Scientific, GibcoTM, catalog number: 52400025 )
  15. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 12657-029 )
  16. Penicillin-streptomycin (Pen-Strep) solution (Biological Industries, catalog number: 03-031-1B )
  17. L-glutamine solution (Biological Industries, catalog number: 03-020-1B )
  18. Gentian violet (Sigma-Aldrich, catalog number: G2039 )
  19. Acetic acid (Sigma-Aldrich, catalog number: ARK2183 )
  20. 0.1 M sodium azide solution (Sigma-Aldrich, catalog number: 0 8591 )
  21. Acetone (Sigma-Aldrich, catalog number: 40289 )
    Note: This product has been discontinued.
  22. Ethanol (Sigma-Aldrich, catalog number: 24103 )
    Note: This product has been discontinued.
  23. Cell dissociation solution (Sigma-Aldrich, catalog number: C5914 )
  24. ddH2O
  25. Coating solution (see Recipes)
  26. Blocking solution with 2% (m/v) BSA (see Recipes)
  27. Complete RPMI medium (see Recipes)
  28. Turk’s solution (see Recipes)
  29. FACS buffer (see Recipes)

Equipment

  1. HeracellTM 150i CO2 incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: 150i CO2 incubator)
  2. Autoclave
  3. Scissors
  4. Forceps
  5. Refrigerator (4° C)
  6. Centrifuge (Eppendorf, model: 5810R )
  7. Centrifuge swing-bucket rotor A-462 4 x 250 ml rectangular buckets (Eppendorf, catalog number: 5810709008 )
  8. Adapters (Eppendorf, catalog number: 5810752000 )
  9. Finnpipette model 4500 single channel pipette:
    1. 0.5-10 μl (Thermo Fisher Scientific, Thermo Scientific, catalog number: FA-10R )
    2. 5-40 μl (Thermo Fisher Scientific, Thermo Scientific, catalog number: FA-40R )
    3. 20-200 μl (Thermo Fisher Scientific, Thermo Scientific, catalog number: FA-200R )
    4. 100-1,000 μl (Thermo Fisher Scientific, Thermo Scientific, catalog number: FA-1000R )
  10. Inverted light microscope (Olympus, model: CHK2-F-GS )
    Note: This product has been discontinued by the manufacturer.
  11. Counting chamber/hemocytometer (Reichert Bright-Line) (Sigma-AIdrich, Bright-LineTM, model: Z359629 )
  12. Flow cytometer (model optional):
    1. MACSQuant VYB instrument (Miltenyi Biotec, model: 130096116 )
    2. FACSCalibur instrument (BD)
    3. FACS LSRII instrument (with all four fixed-aligned lasers) (BD)

Software

  1. CellQuest software
  2. FACSDiva software
  3. FlowJo (Tree Star or V10) or MacsQuant

Procedure

  1. Coating and preparation
    1. Coat tissue culture 6-well plates with 25 µg/ml fibronectin and 2.5 µg/ml CXCL12 overnight at 4 °C. For the coating, it is recommended to prepare a mix of fibronectin and CXCL12 in PBS-/- (coating medium) and to calculate the amount needed so that each well will be covered with 500 µl coating medium. Fibronectin stock (1 mg/ml) and CXCL12 (50 µg/ml) were stored at -20 °C.
    2. After overnight incubation, discard the coating medium gently and wash the plates with 1 ml PBS-/- for a few seconds. Repeat washing 3 times. 
    3. Perform blocking with 2% BSA in PBS-/- for 30 min at room temperature. Blocking solution should cover each well of the plates, and therefore it is recommended to use a minimum of 500 µl blocking solution for each well.
    4. Discard the blocking solution gently and wash plates with 1 ml PBS-/- for a few seconds while tilting the plates slowly. Repeat washing 3 times.
    5. Plates are ready to be seeded with BM mononuclear cells.

  2. Obtaining bone marrow mononuclear cells
    1. Sacrifice desired mice by CO2 euthanasia or cervical dislocation.
    2. Extract the femurs, tibias and pelvises using forceps and sharp scissors. Place the bones on a small dish supplemented with PBS-/-.
    3. Flush total bone marrow cells from the bone cavity with 1-2 ml PBS-/- (per mouse) using a 1 ml syringe and 16 G needle (Figure 1). Obtain single cell suspension by resuspending the cell solution using the same syringe.


      Figure 1. Process of flushing bone marrow out of murine tibia. The red color of the extracted bones indicates the presence of the BM (A). Drilling the needle into the bone and flushing the BM with PBS out of the bone (B) until the bones look white enough (C).

    4. Carefully pipet (very slowly) ficoll to the bottom of each tube with a dilution ratio of 1:2 to the bone marrow cells (i.e., if you have a total of 2 ml bone marrow cells in PBS, pipette 1 ml ficoll to the bottom of the tube) (Figures 2A and 2B).


      Figure 2. Process of MNC isolation from BM. Ficoll is added to the bottom of the tube underneath the suspended BM (A) forming a lower clear ficoll phase and an upper cell suspension phase (B). After centrifugation the two phases are separated by thin white phase consisting of MNCs (C) which is more prominent the more BM is used for the isolation (D).

    5. Centrifuge the cells at 652 x g for 25 min, without a brake (important) at room temperature (mononuclear cells will not be separated if the centrifuge is cold).
    6. Carefully remove the tubes from the centrifuge. At the middle phase a fine white ring-like shape should form, containing the bone marrow mononuclear cells (Figures 2C and 2D)
    7. Carefully, discard the fluids on top of the ring with a pipet, while avoiding touching the mononuclear fraction. Collect the ring of cells with 200 µl pipette tip into a new FACS tube.
    8. Wash the collected mononuclear cells with 2 ml PBS-/- and centrifuge the cells at 452.8 x g for 5 min.
    9. Discard the supernatant and resuspend the cell pellet in 1 ml complete RPMI (see Recipes).
    10. Count the cells by diluting the cells 1:10 with Turk’s solution. Count the cells under the inverted light microscope using a hemocytometer. From one mouse typically one will get 20-40 million mononuclear cells.

  3. Adhesion assay (Figure 3)
    1. Seed bone marrow mononuclear cells at a density of 5 x 106 cells per 1 ml complete RPMI medium.
    2. Allow the cells to adhere to the coated plates for 2 h at 37 °C in an incubator.
    3. Collect non-adherent cells to a new FACS tube by gently rinsing each well with complete RPMI medium. Make sure not to touch the bottom and only add RPMI by placing the pipet tip against the wall of each well.
    4. Gently wash the plates 3 times with PBS-/- by tilting the plates back and forth. Make sure not to touch the bottom and only add PBS-/- by placing the pipet tip against the wall of each well.
    5. To collect the adherent cells, incubate the plates with cell dissociation buffer for 10 min at 37 °C in the incubator.
    6. Monitor the cells under the inverted microscope. If cells are still attached to the bottom, resuspend the cells by using pipet and incubate the plate for additional 5-10 min in the presence of the cell dissociation buffer.
    7. Once the cells are detached from the well, vigorously rinse each well using a pipet and collect the adherent fraction to a new FACS tube. Subsequently, add 1 ml ice cold PBS-/- to each well, rinse thoroughly and collect the fluids into the adherent cell fraction to a different FACS tube.
    8. Monitor the cells under the inverted microscope. If additional cells remained on the plate, vigorously rinse each well again with ice cold PBS-/- and keep collecting the fluids into the adherent cell fraction.
    9. Centrifuge the adherent and nonadherent cell fractions at 452.8 x g, for 5 min.
    10. Discard the supernatant carefully.
    11. Add 100 µl PBS to each pellet. The cells are now ready to be stained and analyzed.

  4. FACS staining and analysis (Figure 4)
    1. Add 1 µl of each lineage antibody (CD8a FITC, CD4 FITC, GR1 FITC, B220 FITC, TER-119 FITC, CD11b FITC) and 2 µl Sca1 PeCy7, cKit APC and EPCR PE antibodies to remaining solution on the bottom of each tube.
      Note: Do not forget to include a negative control in order to be able to distinguish between the autofluorescence and positive signal during the flow cytometry.
    2. Resuspend the pellets by vortexing the tubes briefly.
    3. Leave the samples at 4 °C for 30 min.
      Note: If biotin-coupled antibodies are used, resuspend the cells in 1 ml PBS after the incubation at 4 °C and repeat the steps from C9 until this step. At step D1 add 1 µl of FITC-coupled streptavidin to the remaining solution of each tube.
    4. Resuspend each sample in 1 ml FACS buffer and filter through a 40 µm mesh into a new tube to avoid clots during flow cytometry.
    5. Centrifuge the sample at 452.8 x g for 5 min.
    6. Discard the supernatant carefully, add 300 µl of FACS buffer to each sample and resuspend the cells by vortexing.
    7. Read the samples using a flow cytometer. Cell populations are analyzed with a FACSCalibur instrument with CellQuest software, with a MacsQuant instrument or with a FACS LSRII instrument with FACSDiva software. Please consider that the combination of some fluorophores may cause an overlap in signals within the flow cytometer. Therefore, to set good compensation is mandatory.
    8. Data are analyzed with FlowJo (Tree Star or V10) or MacsQuant according to the gating strategy illustrated in Figure 1 (see Data analysis section as well):
      1. Gate for live cells using forward (FSC) and sideward scatter (SSC).
      2. Gate for lineage negative cells using SSC and the channel for the fluorophore coupled to the lineage antibodies (Figure 5).
      3. Gate for SK cells selecting the c-Kit positive and Sca-1 positive cell population (Figure 5).
      4. Create a histogram overlay displaying the EPCR signals of the LSK population within the negative control, the adherent and nonadherent fractions.


        Figure 3. Diagramm showing steps of adhesion assay procedure (Procedure C)


        Figure 4. Diagramm showing steps of FACS staining procedure (Procedure D)


        Figure 5. Gating strategy of EPCR positive bone marrow LSK cells (adapted from Supp. Figure 5a of Gur-Cohen et al., 2015)

Data analysis

For further analysis information concerning gating strategies and statistical analysis you can consult the article Gur-Cohen et al. (2015) at the following link: http://www.nature.com/nm/journal/v21/n11/full/nm.3960.html.

Recipes

  1. 1x PBS-/-
    Add 50 ml of 10x PBS to 450 ml of dH2O
  2. Coating solution
    1x PBS with 25 µg/ml fibronectin and 2.5 µg/ml CXCL12
  3. Blocking solution with 2% (m/v) BSA
    Add 2 g of BSA per 100 ml 1x PBS
  4. Complete RPMI medium
    88% RPMI 1640 medium
    10% FBS
    1% Pen-Strep solution
    1% L-glutamine solution
  5. Turk’s solution
    50 mg of gentian violet
    5 ml of acetic acid
    495 ml of ddH2O
    Dilute gentian violet in acetic acid and ddH2O
  6. FACS buffer
    Note: Mix this buffer in a plastic bottle.
    450 ml ddH2O
    18 µl sodium azide
    50 ml 10x PBS
    5 ml FBS
  7. Sterilized 24 µm pore-sized nylon mesh
    1. Cut nylon mesh into approx. 3 x 3 pieces
    2. Incubate the meshes for 20 min at room temperature in acetone under the chemical hood
    3. Incubate the meshes for 20 min at room temperature in absolute ethanol
    4. Let the meshes dry over night
    5. Sterilize the meshes by autoclaving them at 121 °C for 60 min

Acknowledgments

This study was supported by the Israel Science Foundation (851/13), the Ernest and Bonnie Beutler Research Program of Excellence in Genomic Medicine and FP7-HEALTH-2010 (CELL-PID 261387) (T.L.) and the DKFZ, Germany.

References

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  28. Wang, X. and Gerdes, H. H. (2015). Transfer of mitochondria via tunneling nanotubes rescues apoptotic PC12 cells. Cell Death Differ 22(7): 1181-1191.

简介

造血干细胞(HSC)由其自我更新的功能定义,并在整个生命中产生所有成熟的血液和免疫细胞类型。大多数HSC在被称为利基的骨髓(BM)的专门的保护性微环境内保持在非运动性静止状态。 HSC通常通过其运动能力与其他成体干细胞区分开来。通过抑制粘附相互作用促进骨髓细胞外基质和血管内皮细胞的物理屏障的移动,这是保留在其BM细胞壁内保留的干细胞所必需的。重要的是,在临床移植后将HSC归巢到BM是重建消融BM的关键的第一步,就像血液恶性肿瘤治疗策略一样。归位过程结束于HSC的选择性访问和锚定到其在BM内的专门的位置。粘附分子是在干细胞移植的情况下增强归巢或减少BM保留以从匹配供体的血液中收集动员的HSC的靶标。在HSC上功能表达并参与其归巢和保留的主要粘附蛋白是整合素α4β1(非常晚的抗原-4; VLA4)。在该方案中,我们引入了针对表达VLA4的鼠骨髓干细胞优化的粘附测定。该测定法在分离表达VLA4的贴壁细胞后,通过流式细胞术与HSC富集细胞表面标记物定量粘附的HSC。

背景 HSCs主要保留在BM中,并通过与其微环境(niche)的粘合相互作用来调节。以这种方式,HSC保持在非运动性静止状态,保护它们免受DNA损伤代理(Boulais和Frenette,2015; Mendelson和Frenette,2014; Miyamoto等人,2011; Morrison and Scadden ,2014)。 HSC的定义属性是其持久地重新填充移植受体的辐射BM的功能能力,这需要其归巢,自我更新和发育潜力(Gur-Cohen等人,2016)。由于粘附引起细胞内信号通路的激活,相互作用的类型可以反映细胞的发育状态和行为(Sugiyama等人,2006)。粘附试验是区分粘合剂和非粘性细胞的方法。在该方案中,我们在静态条件下引入细胞粘附分析,分离VLA4表达的粘附细胞与非粘附细胞,通过FACS分析进行定量。
 在小鼠中,造血干细胞和祖细胞(HSPC)富集在缺乏谱系标记物(Lin; CD8a,CD4,GR1,B220,TER-119,CD11b)的群体中,并表达c-Kit(K)和Sca -1(S)。因此,这些细胞也称为Lin-Sca-1 + c-Kit + (LSK)细胞(Adolfsson等人,2001; Okada等人,1991; Spangrude等人,1988)。 EPCR(内皮蛋白C受体)已经在各种其他组织中被鉴定为干细胞标记(Balazs等人,2006; Iwasaki等人,2010; Kent 等人,2009; Ramalho-Santos等人,2002; Wang和Gerdes,2015)。
 粘附分子在BM和血液循环中保留和排出这些HSC中起主要作用。 VLA4是纤连蛋白和VCAM-1的受体,并且由大多数白细胞以及一些非造血细胞(Hemler等人,1990)表达,而其在小鼠中的表达更高与EPCR阴性祖细胞和循环LT-HSC(Gur-Cohen等人,2015)相比,BM EPCR + LT-HSC。长期以来,已提出LT-HSC的VLA4表达对于人BM微环境中干细胞的结合和分离可能是重要的。通过中和抗体抑制VLA4或VCAM-1结合导致HSPC从BM的动员到小鼠和灵长类动物的血液循环(Craddock等人,1997; Papayannopoulou等人,这与VLA4对于BM(Papayannopoulou等人,1995; Papayannopoulou和Scadden,2008)中CXCL12 / CXCR4介导的LT-HSC静止保留至关重要的概念是一致的, 。除了HSPC BM保留之外,VLA4对于鼠HSPC BM归巢也是必需的(Papayannopoulou和Craddock,1997)。 VLA4具有与其亲和性状态相关的不同构象(Alon等人,1995; Chen等人,1999; Feigelson等人。
,2001),其受二价阳离子和内向外信号的影响(Chigaev等人,2003; Chigaev等人,2011)。大多数VLA4亲和性内向外信号传导是由G蛋白偶联受体介导的(Laudanna等人,2002; Chigaev等人,2008; Arnaout et al。,2007)。此外,细胞内一氧化氮(NO)的升高显示导致cGMP介导的VLA4亲和力的抑制(Chigaev等人,2011)。我们以前已经显示了两种不同的途径,即aPC-EPCR-PAR1和凝血酶-PA1轴,其分别上调和下调NO水平。因此,这些途径影响许多细胞内分子,包括Cdc42,CXCR4和VLA4,导致HSPC的保留或动员(Gur-Cohen等人,2015)。如Gur-Cohen等人(2015)所述,我们在此提出了用于EPCR 干细胞的VLA4介导的粘附测定作为预测LT-HSC保留的有力工具他们的骨髓利基的潜力。

关键字:极晚期抗原4, 整合素α4β1, 粘附测定, 内皮蛋白C受体(EPCR), 长期再生造血干细胞(LT-HSC), 流式细胞术

材料和试剂

  1. 组织培养6孔板(Corning,Costar ®,目录号:3516)
  2. Nunc TM 8.8cm 2培养皿(Thermo Fisher Scientific,Thermo Scientific TM,目录号:153066)
  3. 1毫升滑头Sub-Q注射器与一次性26 G x 5/8英寸针(BD,目录号:309597)
  4. 高压灭菌吸管技巧:
    1. 200-1,000μl蓝,吉尔森提示(Greiner Bio One,目录号:740290)
    2. 200μl通用尖端(Neptune Scientific,目录号:2100.N)
    3. 0.5-10μl吸头(Corning,Axygen ®,目录号:T-300)
  5. FACS管(Corning,Falcon ®,型号:352054)
  6. 灭菌40μm孔径尼龙网(Sinun Tech,目录号:Plymer Screends)(参见食谱)
  7. 小鼠
  8. 纤连蛋白(Sigma-Aldrich,目录号:F0895)
  9. 人CXCL12(人类SDF-1α)(繁殖,目录号:RKP48061)或(PeproTech,Rocky Hill,NJ,USA)
  10. BSA(Sigma-Aldrich,目录号:A9647)
  11. Lymphoprep TM Ficoll(STEMCELL Technologies,目录号:07861)
  12. 抗体:
    1. EPCR PE(Affymetrix,eBioscience,目录号:12-2012-82)或EPCR PerCP-eFluor 710(Affymetrix,eBioscience,目录号:46-2012-80)或EPCR生物素(Affymetrix,eBioscience,目录号:13-2012 -82)与链霉亲和素-PE(BioLegend,目录号:405203)结合使用
    2. Sca-1 PE(BioLegend,目录号:108108)或Sca-1 PE/Cy7(BioLegend,目录号:108114
    3. c-Kit APC(BioLegend,目录号:105812)
    4. 谱系抗体
      注意:这是一组针对以下列出的抗原i-vi的抗体,其是细胞谱系标记。在小鼠中,这些标记物不会发生在干细胞和祖细胞上
      1. CD8a FITC(BioLegend,目录号:100706)
      2. CD4 FITC(BioLegend,目录号:100406)
      3. GR1 FITC(BioLegend,目录号:108406)
      4. B220 FITC(BioLegend,目录号:103206)
      5. TER-119 FITC(BioLegend,目录号:116206)
      6. CD11b FITC(BioLegend,目录号:101206)
      7. 或血清鸡尾酒生物素(Miltenyi Biotec,目录号:130-092-613)与链霉抗生物素蛋白FITC(BioLegend;目录号:405202)结合
        注意:用于实验的荧光团的组合需要根据实验需求进行调整。
  13. 1x Dulbecco的磷酸盐缓冲盐水,不含钙和镁(PBS(PBS),见Recipes)(Biological Industries,目录号:02-023-5A)
  14. Roswell公园纪念研究所(RPMI)1640培养基([+] 300mg/L L-谷氨酰胺,[+] 25mM HEPES)(Thermo Fisher Scientific,Gibco TM,目录号:52400025)
  15. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco ,目录号:12657-029)
  16. 青霉素 - 链霉素(Pen-Strep)溶液(Biological Industries,目录号:03-031-1B)
  17. L-谷氨酰胺溶液(Biological Industries,目录号:03-020-1B)
  18. 龙胆紫(Sigma-Aldrich,目录号:G2039)
  19. 乙酸(Sigma-Aldrich,目录号:ARK2183)
  20. 0.1M叠氮化钠溶液(Sigma-Aldrich,目录号:08591)
  21. 丙酮(Sigma-Aldrich,目录号:40289)
    注意:本产品已停产。
  22. 乙醇(Sigma-Aldrich,目录号:24103)
    注意:本产品已停产。
  23. 细胞解离溶液(Sigma-Aldrich,目录号:C5914)
  24. ddH 2 O
  25. 涂层溶液(见配方)
  26. 用2%(m/v)BSA封闭溶液(参见食谱)
  27. 完成RPMI培养基(见食谱)
  28. 土耳其语的解决方案(见食谱)
  29. FACS缓冲液(参见食谱)

设备

  1. Heacell孵化器(Thermo Fisher Scientific,Thermo Scientific ,型号:150i CO 2孵育器)
  2. 高压灭菌器
  3. 剪刀
  4. 镊子
  5. 冰箱(4°C)
  6. 离心机(Eppendorf,型号:5810R)
  7. 离心机摇摆转子A-462 4×250毫升矩形桶(Eppendorf,目录号:5810709008)
  8. 适配器(Eppendorf,目录号:5810752000)
  9. Finnipipette型号4500单通道移液器:
    1. 0.5-10μl(Thermo Fisher Scientific,Thermo Scientific,目录号:FA-10R)
    2. 5-40μl(Thermo Fisher Scientific,Thermo Scientific,目录号:FA-40R)
    3. 20-200μl(Thermo Fisher Scientific,Thermo Scientific,目录号:FA-200R)
    4. 100-1,000μl(Thermo Fisher Scientific,Thermo Scientific,目录号:FA-1000R)
  10. 倒置光学显微镜(Olympus,型号:CHK2-F-GS)
    注意:本产品已经被制造商停产。
  11. 计数室/血细胞计数器(Reichert Bright-Line)(Sigma-AIdrich,Bright-Line TM,型号:Z359629)
  12. 流式细胞仪(型号可选):
    1. MACSQuant VYB仪器(Miltenyi Biotec,型号:130096116)
    2. FACSCalibur仪器(BD)
    3. FACS LSRII仪器(全部四个固定校准激光器)(BD)

软件

  1. CellQuest软件
  2. FACSDiva软件
  3. FlowJo(Tree Star或V10)或MacsQuant

程序

  1. 涂层和制备
    1. 在4℃下,将具有25μg/ml纤连蛋白和2.5μg/ml CXCL12的涂层组织培养6孔板过夜。对于涂层,建议在PBS -/- (涂层介质)中制备纤连蛋白和CXCL12的混合物,并计算所需的量,以使每孔用500μl涂层培养基覆盖。将纤连蛋白原(1mg/ml)和CXCL12(50μg/ml)储存于-20℃。
    2. 孵育过夜后,轻轻丢弃涂层培养基,并用1 ml PBS /平板洗涤平板几秒钟。重复洗涤3次。
    3. 在室温下,用PBS 中的2%BSA进行30分钟的封闭。封闭溶液应覆盖板的每个孔,因此建议每个孔使用至少500μl的封闭溶液。
    4. 轻轻倒掉封闭溶液,并用1 ml PBS /洗涤板数秒钟,同时缓慢倾斜板。重复洗涤3次。
    5. 板准备接种BM单核细胞。

  2. 获得骨髓单核细胞
    1. 通过CO 2安乐死或颈椎脱位牺牲所需的小鼠。
    2. 使用镊子和锋利的剪刀提取股骨,胫骨和骨盆。将骨头放在补充有PBS -/- 的小盘子上。
    3. 使用1ml注射器和16G针头(图1),用1-2ml PBS(每只小鼠)从骨腔中冲洗总骨髓细胞。通过使用相同的注射器重悬细胞溶液来获得单细胞悬液。


      图1.从鼠胫骨冲洗骨髓的过程。提取的骨骼的红色表示存在BM(A)。将针头钻入骨头并用PBS将骨骼(B)从骨头(B)中冲洗,直到骨头看起来足够白(C)。

    4. 仔细移取(非常慢)的每个管的底部,稀释比为1:2与骨髓细胞(如,如果您在PBS中总共有2ml骨髓细胞,将1毫升ficoll移液到管底部)(图2A和2B)

      图2. MNC从BM中分离的过程将Ficoll加入到悬浮的BM(A)下面的管底部,形成较低的澄清Ficoll相和上细胞悬浮相(B)。离心后,两相由MNC(C)组成的薄白相分离,BM分离更为显着(D)。

    5. 将细胞以652 x g离心25分钟,在室温下无刹车(重要)(如果离心机冷却,则单核细胞不会分离)。
    6. 小心地从离心机中取出管子。在中期,应形成细小的白色环状形状,含有骨髓单核细胞(图2C和2D)
    7. 小心地,用移液管丢弃环上方的液体,同时避免接触单核级分。用200μl移液器吸头将细胞环收集到新的FACS管中
    8. 用2ml PBS洗涤收集的单核细胞,并以452.8×g离心细胞5分钟。
    9. 丢弃上清液并将细胞沉淀重悬于1ml完整的RPMI中(参见食谱)。
    10. 用Turk的溶液稀释细胞1:10计数细胞。使用血细胞计数器在倒置光学显微镜下计数细胞。一只老鼠通常会得到20-40万个单核细胞。

  3. 粘附试验(图3)
    1. 每1ml完全RPMI培养基的密度为5×10 6个细胞的种子骨髓单核细胞。
    2. 允许细胞在37℃下在培养箱中粘附到包被的板上2小时。
    3. 通过用完整的RPMI培养基轻轻冲洗每个孔,将非贴壁细胞收集到新的FACS管中。确保不要触摸底部,只能通过将移液管针头放在每个孔的墙壁上来添加RPMI。
    4. 通过前后倾斜板轻轻洗板3次,PBS -/- 确保不要触摸底部,只能通过将移液管针头放在每口井的墙壁上来添加PBS -/-
    5. 为了收集贴壁细胞,在培养箱中37℃孵育平板与细胞解离缓冲液10分钟。
    6. 在倒置显微镜下监测细胞。如果细胞仍然连接到底部,则通过使用移液管重悬细胞,并在细胞解离缓冲液存在下孵育平板另外5-10分钟。
    7. 一旦细胞从孔中分离出来,用移液管大力冲洗每个孔,并将粘附部分收集到新的FACS管中。随后,向每个孔中加入1ml冰冷PBS,将其彻底冲洗,并将流体收集到贴壁细胞部分中至不同的FACS管中。
    8. 在倒置显微镜下监测细胞。如果额外的细胞残留在平板上,用冰冷的PBS重新冲洗每个孔,并继续将流体收集到贴壁细胞部分中。
    9. 以452.8×g离心粘附和非贴壁细胞级分5分钟。
    10. 仔细舍弃上清液。
    11. 向每个颗粒加入100μlPBS。细胞现在可以被染色和分析。

  4. FACS染色和分析(图4)
    1. 在每个管的底部添加1μl每个谱系抗体(CD8a FITC,CD4 FITC,GR1 FITC,B220 FITC,TER-119 FITC,CD11b FITC)和2μlSca1 PeCy7,cKit APC和EPCR PE抗体至剩余溶液。
      注意:不要忘记包括阴性对照,以便能够在流式细胞仪中区分自发荧光和阳性信号。
    2. 简单地涡旋管重新悬浮颗粒。
    3. 将样品放在4°C 30分钟 注意:如果使用生物素偶联的抗体,则在4℃下孵育后将细胞重悬于1ml PBS中,并重复步骤C9至该步骤。在步骤D1,向每个管的剩余溶液中加入1μlFITC偶联的链霉亲和素。
    4. 将每个样品重悬于1 ml FACS缓冲液中,并通过40μm筛网过滤到新管中,以避免流式细胞术中出现凝块。
    5. 以452.8×g离心样品5分钟。
    6. 仔细弃去上清液,向每个样品中加入300μlFACS缓冲液,并通过涡旋重悬细胞
    7. 使用流式细胞仪读取样品。用具有CellQuest软件的FACSCalibur仪器,MacsQuant仪器或具有FACSDiva软件的FACS LSRII仪器分析细胞群体。请考虑一些荧光团的组合可能会导致流式细胞仪内的信号重叠。因此,设定好的补偿是强制性的
    8. 数据使用FlowJo(Tree Star或V10)或MacsQuant根据图1所示的门控策略进行分析(参见数据分析部分):
      1. 使用正向(FSC)和侧向散射(SSC)的活细胞门。
      2. 使用SSC的谱系阴性细胞的门和与谱系抗体偶联的荧光团的通道(图5)。
      3. SK细胞门选择c-Kit阳性和Sca-1阳性细胞群(图5)
      4. 创建一个直方图覆盖,显示阴性对照中LSK群体的EPCR信号,粘附和非粘附级数。


        图3.显示粘附测定程序步骤的图(步骤C)


        图4.显示FACS染色程序步骤(方法D)的图示


        图5. EPCR阳性骨髓LSK细胞的门控策略(改编自Gur-Cohen等人,2015年的Supp图5a)

数据分析

有关门控策略和统计分析的进一步分析信息,您可以参考Gur-Cohen等人的文章。 (2015)在以下链接: http://www.nature.com/nm/journal/v21/n11/full/nm.3960.html

食谱

  1. 1x PBS -/-
    加入50毫升10倍的PBS至450毫升的dH 2 O -/-
  2. 涂层溶液
    1×PBS,25μg/ml纤连蛋白和2.5μg/ml CXCL12
  3. 2%(m/v)BSA阻塞溶液
    每100ml 1x PBS加入2μgBSA
  4. 完成RPMI培训
    88%RPMI 1640培养基
    10%FBS
    1%Pen-Strep溶液
    1%L-谷氨酰胺溶液
  5. 土耳其的解决方案
    50毫升龙胆紫色
    5毫升乙酸
    495毫升的ddH 2 O -/- 在乙酸和ddH 2 O中稀释龙胆紫 -
  6. FACS缓冲区
    注意:将此缓冲液混合在塑料瓶中。
    450毫升ddH 2 O -/- 18μl叠氮化钠
    50ml 10x PBS
    5 ml FBS
  7. 灭菌的24μm孔径尼龙网
    1. 将尼龙网切成约3 x 3件
    2. 在化学引擎罩下,在丙酮中室温孵育20分钟
    3. 在室温下用无水乙醇培养网眼20分钟
    4. 让网格过夜干净
    5. 通过在121℃高压灭菌60分钟来灭菌网眼

致谢

这项研究得到了以色列科学基金会(851/13),欧内斯特和邦妮·贝特勒基因组医学优秀研究计划和FP7-HEALTH-2010(CELL-PID 261387)(T.L.)和德国DKFZ的支持。

参考文献

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引用:Avci, S., Gur-Cohen, S., Avemaria, F. and Lapidot, T. (2017). Adhesion Assay for Murine Bone Marrow Hematopoietic Stem Cells. Bio-protocol 7(4): e2135. DOI: 10.21769/BioProtoc.2135.
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