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Isolation of Human Blood Progenitor and Stem Cells from Peripheral Blood by Magnetic Bead
磁珠法从外周血液中分离人血前体细胞和干细胞   

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Abstract

The antigen CD34 is a well-known marker present on human progenitor and stem cells. This protocol explains the isolation of CD34+ cells from peripheral blood using magnetic bead separation technique. The approximate abundance of CD34+ cells in blood is 0.1% of mononuclear cells.

Keywords: Hematopoiesis(造血), Progenitors(脑细胞前体), Isolation(隔离)

Materials and Reagents

  1. CD34+ cells
  2. Peripheral blood sample (at least 50 ml)
  3. Dextran solution (Sigma-Aldrich, catalog number: D1037-500G )
  4. PBS (Life Technologies, Invitrogen™, catalog number: 14190-094 )
  5. Ficoll human (PAA Laboratories GmbH, catalog number: P04-60500 )
  6. Fetal calf serum (FCS) (Hyclone, catalog number: SV30160.03 )
  7. CD34 MicroBeads and FcR blocking reagent (Miltenyi Biotec, catalog number: 130-046-702 )
  8. APC mouse anti-human CD34 antibody (BD Biosciences, catalog number: 555824 )
  9. EDTA
  10. 2% dextran solution
  11. 1 L 2% dextran solution (see Recipes)

Equipment

  1. Centrifuges
  2. Auto MACS Pro separator (Miltenyi Biotec)
  3. 30 μm nylon mesh (Miltenyi Biotec, catalog number: 130-041-407 )
  4. Tissue culture hood
  5. Flow cytometer
  6. Vacuum filter unit (22 μm, GP Millipore Express PLUS membrane)

Procedure

  1. Peripheral blood mononuclear cells (PBMCs) separation
    1. Under tissue culture hood, add equal volume of 2% dextran solution and blood sample and incubate it at room temperature for 45 min (elimination of majority of erythrocytes).
    2. Collect the supernatant and centrifuge at 1,600 rpm (ROTANA 460RF, rotor 5624 Hettich) for 10 min.
    3. Discard the supernatant and re-suspend the pellet, containing lymphocytes and granulocytes, in 10 ml PBS with 0.1% EDTA.
    4. Add 7.5 ml ficoll solution to a suitable centrifuge tube and carefully top it with 10 ml cell suspension from step A3.
    5. Centrifuge at 2,200 rpm for 20 min.
    6. After centrifugation there are three phases: Upper one containing plasma and platelets. The whitish layer containing the mononuclear cells and the lower phase and pellet containing the granulocytes.
    7. Discard upper aqueous phase containing plasma and collect the whitish layer containing PBMCs (around 2 ml) and re-suspend it in 50 ml of PBS with 0.1% EDTA.
    8. Count the PBMCs.
    9. Centrifuge the PBMCs at 1,200 rpm for 10 min and re-suspend 1 x 108 cells in 300 μl of PBS with 0.1% EDTA. If you have 5 x 107 cells, you need to resuspend in 150 μl of PBS with 0.1% EDTA.

  2. CD34 magnetic labeling
    1. Add CD34 MicroBeads and FcR blocking reagent (100 μl each for 1 x 108 cells) to 300 μl of PBMCs suspension. The FcR blocking reagent is used to avoid non specific labeling.
    2. Mix well and incubate at 4 °C for 30 min (or overnight with 30% FCS).

  3. Magnetic separation with auto MACS separator
    1. Add 10 ml PBS 0.1% EDTA to magnetic beads labeled cells and centrifuge them at 1,200 rpm for 10 min.
    2. Discard supernatant and re-suspend the cells in PBS with 0.1% EDTA (500 μl for 1x 108 cells).
    3. Filter the cells using 30 μm nylon mesh to exclude the cell clumps and rinse the tube and filter with additional 500 μl PBS with 0.1% EDTA to collect maximum cells.
    4. Pass the cells through auto MACS separator as explained in user manual (CD34 MicroBead kit human).
    5. After separation, collect CD34+ cells and determine cell number.
    6. Wash the cells with 5-10 ml PBS 0.1% EDTA at 1,200 rpm for 10 min at room temperature.
    7. Once washed re-suspend cells in appropriate cell culture medium or freeze them for later use.

  4. CD34+ cells purity assessment
    1. Take 10 x 103 cells from step C 4 and re-suspend them in 100 μl of PBS.
    2. Add 1 μl of APC-conjugated mouse anti-human CD34 antibody.
    3. Incubate for 15-20 min at 4 °C in dark.
    4. Wash the cells with 500 μl PBS at 1,200 rpm for 10 min.
    5. Discard the supernatant and re-suspend the cells in 300 μl PBS.
    6. Pass them through a flow cytometer and analyze the cells for APC labeling excluding dead cells and debris using scatter signals.

Recipes

  1. 1 L 2% dextran solution
    Add 9 g NaCl and 20 g dextran to beaker containing 800 ml deionized H2O
    Dissolve NaCl and dextran using magnetic stirrer and magnetic stirrer bar (approx. 2 h)
    Add deionized H2O to make up the volume to 1 L
    Under tissue culture hood, filter the 2% dextran solution using 22 μm pore vacuum filter
    Conserve the solution at 4 °C

Acknowledgments

The protocol was previously published in Nakatake et al. (2012). This work was supported by grants from ” Association pour la Recherche sur le Cancer (projet libre 2012), Agence Nationale de la Recherche, programme Jeunes Chercheuses et Jeunes Chercheurs, Laboratory of Excellence Globule Rouge-Excellence is funded by the program “Investissements d’avenir.” HS was supported by fellowships from la Ligue Nationale Contre le Cancer.

References

  1. Nakatake, M., Monte-Mor, B., Debili, N., Casadevall, N., Ribrag, V., Solary, E., Vainchenker, W. and Plo, I. (2012). JAK2(V617F) negatively regulates p53 stabilization by enhancing MDM2 via La expression in myeloproliferative neoplasms. Oncogene 31(10): 1323-1333.

简介

抗原CD34是存在于人祖细胞和干细胞上的公知标记。 该方案解释了使用磁珠分离技术从外周血中分离CD34 +细胞。 血液中CD34 +细胞的大致丰度为0.1%的单核细胞。

关键字:造血, 脑细胞前体, 隔离

材料和试剂

  1. CD34 + 单元格
  2. 外周血样(至少50ml)
  3. 葡聚糖溶液(Sigma-Aldrich,目录号:D1037-500G)
  4. PBS(Life Technologies,Invitrogen TM,目录号:14190-094)
  5. Ficoll人(PAA Laboratories GmbH,目录号:P04-60500)
  6. 胎牛血清(FCS)(Hyclone,目录号:SV30160.03)
  7. CD34 MicroBeads和FcR阻断试剂(Miltenyi Biotec,目录号:130-046-702)
  8. APC小鼠抗人CD34抗体(BD Biosciences,目录号:555824)
  9. EDTA
  10. 2%葡聚糖溶液
  11. 1 L 2%葡聚糖溶液(见配方)

设备

  1. 离心机
  2. Auto MACS Pro分离器(Miltenyi Biotec)
  3. 30μm尼龙网(Miltenyi Biotec,目录号:130-041-407)
  4. 组织培养罩
  5. 流式细胞仪
  6. 真空过滤器单元(22μm,GP Millipore Express PLUS膜)

程序

  1. 外周血单核细胞(PBMC)分离
    1. 在组织培养罩下,加入等体积的2%葡聚糖溶液和血液样品,并在室温下孵育45分钟(消除大多数红细胞)。
    2. 收集上清液并以1,600rpm(ROTANA 460RF,转子5624Hettich)离心10分钟。
    3. 弃去上清液并将含有淋巴细胞和粒细胞的沉淀重悬于10ml含0.1%EDTA的PBS中。
    4. 将7.5 ml ficoll溶液加入到合适的离心管中,并用10ml来自步骤A3的细胞悬浮液小心地顶部
    5. 以2200rpm离心20分钟。
    6. 离心后有三个阶段:上层包含血浆和血小板。 白色层含有单核细胞和下层相,颗粒含有粒细胞
    7. 弃去含有血浆的上层水相,收集含有PBMC的白色层(约2ml),并将其重悬于50ml含0.1%EDTA的PBS中。
    8. 计数PBMC。
    9. 将PBMC以1200rpm离心10分钟,并将300μl含有0.1%EDTA的PBS中的1×10 8个细胞重悬浮。 如果您有5 x 10 7 细胞,则需要重悬于150μl含0.1%EDTA的PBS中。

  2. CD34磁性标记
    1. 向300μlPBMC悬浮液中加入CD34 MicroBeads和FcR阻断试剂(各1×10 8个细胞各100μl)。 FcR阻断试剂用于避免非特异性标记。
    2. 充分混合并在4℃孵育30分钟(或用30%FCS过夜)

  3. 磁分离与自动MACS分离器
    1. 加入10 ml PBS 0.1%EDTA磁珠标记细胞,并在1,200 rpm离心10分钟
    2. 弃去上清液并将细胞重悬于含有0.1%EDTA的PBS中(500μl,对于1×10 8个细胞)。
    3. 使用30μm尼龙网过滤细胞以排除细胞团,并冲洗管,并用额外的500μl含0.1%EDTA的PBS过滤,收集最大细胞。
    4. 将细胞通过自动MACS分离器,如用户手册(CD34 MicroBead Kit human)中所述。
    5. 分离后,收集CD34 + 细胞并测定细胞数
    6. 用5-10ml PBS 0.1%EDTA在室温下以1,200rpm洗涤细胞10分钟
    7. 一旦洗涤,将细胞重悬在适当的细胞培养基中或将其冻结以备后用

  4. CD34 + 细胞纯度评估
    1. 取来自步骤C 4的10×10 3个细胞,并将它们重悬在100μl的PBS中。
    2. 加入1μl的APC结合的小鼠抗人CD34抗体
    3. 在4℃黑暗中孵育15-20分钟。
    4. 用500μlPBS以1,200 rpm洗涤细胞10分钟。
    5. 弃去上清液并将细胞重悬在300μlPBS中
    6. 使它们通过流式细胞仪,并使用散射信号分析细胞用于APC标记,不包括死细胞和碎片

食谱

  1. 1L 2%葡聚糖溶液
    将9g NaCl和20g葡聚糖加入到含有800ml去离子H 2 O的烧杯中
    使用磁力搅拌器和磁力搅拌棒(约2小时)溶解NaCl和葡聚糖 加入去离子H 2 O以使体积达到1L
    在组织培养罩下,使用22μm孔真空过滤器
    过滤2%葡聚糖溶液 在4℃保存溶液

致谢

该协议先前已在Nakatake等人(2012)中公布。这项工作得到了来自"协会资助癌症(Projet libre 2012),Agence Nationale de la Recherche,计划Jeunes Chercheuses和Jeunes Chercheurs,卓越实验室Globule Rouge-Excellence的资金资助的方案"Investissements d' avenir"。HS得到了La Ligue Nationale Contrele Cancer的奖学金的支持。

参考文献

  1. Nakatake,M.,Monte-Mor,B.,Debili,N.,Casadevall,N.,Ribrag,V.,Solary,E.,Vainchenker,W.and Plo, JAK2(V617F)通过在骨髓增生性肿瘤中通过La表达增强MDM2来负调节p53稳定。 Oncogene 31(10):1323-1333。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Hasan, S. and Plo-Azevedo, I. (2012). Isolation of Human Blood Progenitor and Stem Cells from Peripheral Blood by Magnetic Bead . Bio-protocol 2(21): e281. DOI: 10.21769/BioProtoc.281.
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