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In vivo BrdU Incorporation Assay for Murine Hematopioetic Stem Cells

小鼠造血干细胞的体内BrdU掺入标记试验

Ningfei An Ningfei An
Yubin Kang Yubin Kang

发布: 2013年11月05日第3卷第21期 DOI: 10.21769/BioProtoc.960 浏览次数: 17050

评审: Lin FangAnonymous reviewer(s)

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Cover of Stem Cells, featuring study using the protocol.

Jun 2013

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Abstract

Bromodeoxyuridine (BrdU) is a thymidine analog that is incorporated into DNA during the S-phase of the cell cycle. As such, BrdU incorporation can be used to quantify the number of cells that are in S-phase in the time period during which BrdU is available. The following protocol describes an in vivo BrdU incorporation assay as a measure of cell proliferation in adult murine hematopioetic stem cells (HSCs). Specifically, BrdU incorporation was analyzed for long-term HSCs (LT-HSCs, Lin-Sca-1+c-Kit+CD34-CD135-), Short-term HSCs (ST-HSCs, Lin-Sca-1+c-Kit+CD34+CD135-) and multipotent progenitors (MPPs, Lin-Sca-1+c-Kit+CD34+CD135+) population.

Materials and Reagents

  1. Mouse
  2. BrdU
  3. RPMI1640
  4. Fetal Bovine Serum (FBS)
  5. Potassium bicarbonate
  6. Ammonium chloride
  7. EDTA
  8. BSA
  9. Lineage Cell Depletion Kit (including Biotin-Antibody Cocktail and Anti-Biotin MicroBeads) (Miltenyi Biotec, catalog number: 130-090-858 )
  10. BD Cytofix/Cytoperm Buffer (Becton, Dickinson and Company, catalog number: 554714 )
  11. BD Perm/Wash Buffer
  12. DNase (included in FITC BrdU Flow Kit) (Becton, Dickinson and Company, catalog number: 559619 )
  13. DPBS without calcium, magnesium (diluted from 10x DPBS) (Hyclone, catalog number: SH 30258.01 )
  14. Antibodies:
    PE-conjugated-Sca-I (Becton, Dickinson and Company, catalog number: 553336 )
    APC-conjugated-c-Kit (Becton, Dickinson and Company, catalog number: 553356 )
    PerCP-eFluor 710-conjugated-CD135 (eBioscience, catalog number: 46-1351-80 )
    eFluor 450-conjugated CD34 (eBioscience, catalog number: 48-0341-80 )
    BrdU-FITC (included in FITC BrdU Flow Kit) (Becton, Dickinson and Company, catalog number: 559619)
  15. Buffer A (see Recipes)
  16. Red blood cell lysis buffer (see Recipes)
  17. Staining buffer (see Recipes)

Equipment

  1. Small scissors and forceps
  2. 60 mm tissue culture dish
  3. 23 G needle
  4. 3 cc syringe
  5. 15 ml centrifuge tube
  6. Cell strainer (Becton, Dickinson and Company, catalog number: 352340 )
  7. Hemacytometer
  8. MACS MS column (Miltenyi Biotec, catalog number: 130-042-201 )
  9. MiniMASC separator (Miltenyi Biotec, catalog number: 130-042-102 )
  10. Centrifuge
  11. BD LSRFortessa Analytical Flow Cytometer

Procedure

English
中文翻译

文章信息

版权信息

© 2013 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

An, N. and Kang, Y. (2013). In vivo BrdU Incorporation Assay for Murine Hematopioetic Stem Cells. Bio-protocol 3(21): e960. DOI: 10.21769/BioProtoc.960.

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分类

干细胞 > 成体干细胞 > 造血干细胞

细胞生物学 > 细胞活力 > 细胞增殖

分子生物学 > DNA > DNA 标记

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