Isolation of Prolymphocytes from Bone Marrow and Fetal Liver

Taku Kouro1, Takafumi Yokota1, Robert Welner1, Paul W. Kincade1

1 Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 22F.1
DOI:  10.1002/0471142735.im22f01s66
Online Posting Date:  May, 2005
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Abstract

Isolation of the lymphoid progenitors in their earliest stage of development is indispensable for understanding when and how hematopoietic cell lineages are committed. Recently developed immunofluorescent labeling and sorting has made it possible to identify and isolate rare progenitors of lymphocytes. In this unit, protocols for isolating lymphoid progenitors from three different sources are provided. These protocols are based on stepwise purification consisting of magnetic and fluorescence‐activated cell sorting techniques, optimized according to the nature of progenitors in each organ. Isolated progenitors may be analyzed in the lymphocyte differentiation cultures described in the following units and are useful for in vivo transfer experiments or microarray assays for gene expression.

Keywords: B lymphocyte; progenitors; cell sorting; bone marrow; fetal liver

     
 
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Table of Contents

  • Section F: Differentiation of Special Populations of Stem and Progenitor Cells
  • Basic Protocol 1: Isolation of Prolymphocytes from Adult Mouse Bone Marrow
  • Basic Protocol 2: Isolation of Early Lymphoid Progenitors from Fetal Liver
  • Basic Protocol 3: Isolation of Human Lymphohematopoietic Progenitors
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Prolymphocytes from Adult Mouse Bone Marrow

  Materials
  • Mice (6 to 20 weeks old)
  • 70% ethanol
  • Sterile wash buffer: combine 500 ml PBS ( appendix 2A) with 15 ml fetal bovine serum (FBS) and filter‐sterilize using a 0.22‐µm filter (store up to 1 year at 4°C)
  • Purified, unlabeled rat monoclonal antibodies directed against lineage markers (BD Pharmingen): anti‐CD45R(B220), anti‐CD11b(Mac‐1), anti‐erythroid (TER‐119), anti‐CD3, and anti‐Ly6G(Gr‐1)
  • 5× anti‐rat IgG‐coated microbeads for MACS separation system (Miltenyi Biotec)
  • MACS buffer (see recipe), ice‐cold
  • Labeled lineage antibodies (purchase from BD Pharmingen unless otherwise indicated): FITC‐anti‐Ly6G(Gr‐1), FITC‐anti‐CD11b(Mac‐1), FITC‐TER‐119 (purchase from eBioscience), FITC‐anti‐CD19, FITC‐anti‐CD45R(B220), FITC‐anti‐CD3ɛ, FITC‐anti‐CD49b(DX5), PE‐anti‐CD127(IL‐7Rα), biotinylated anti‐CD135(Flk‐2/Flt) (biotinylate purified antibody from BD Pharmingen using sulfo‐NHS‐biotin from Pierce, according to manufacturer's instructions), APC‐anti‐CD117(c‐kit)
  • Streptavidin‐PE–Texas Red (Caltag)
  • Petri dishes, sterile
  • Dissecting instruments: forceps and scissors
  • 50‐ml conical tubes
  • Syringe with 26‐G needle
  • 70‐µm nylon‐mesh cell strainer (Falcon cat. no. 352350)
  • MACS negative selection (LD) columns (Miltenyi Biotec)
  • Midi MACS separation magnet and magnet stand (Miltenyi Biotec)
  • 15‐ml conical polypropylene tubes
  • Fluorescence‐activated cell sorter with 2‐laser, 4‐color capacity
  • Additional reagents and equipment for mouse euthanasia (unit 1.8), preparing bone marrow cell suspensions (unit 6.4), cell counting ( appendix 3B), and flow cytometry (Chapter 5)

Basic Protocol 2: Isolation of Early Lymphoid Progenitors from Fetal Liver

  Materials
  • Pregnant mice, gestation day 13 or 14
  • D‐PBS with calcium and magnesium ( appendix 1U)
  • Sterile wash buffer: combine 500 ml PBS ( appendix 2A) with 15 ml fetal bovine serum (FBS) and filter‐sterilize using a 0.22‐µm filter (store up to 1 year at 4°C)
  • Labeled antibodies (purchase from BD Pharmingen unless otherwise indicated): FITC‐TER‐119 (purchase from eBioscience), FITC‐anti‐CD19 (optional), PE‐anti‐Sca1, APC‐anti‐CD117(c‐kit)
  • 1000× 7‐aminoactinomycin D (7‐AAD) stock solution (see recipe)
  • Dissecting instruments: small scissors and fine forceps
  • Microscope slides
  • 70‐µm nylon‐mesh cell strainer (Falcon cat no. 352350)
  • Purified (unlabeled) rat monoclonal antibodies (BD Pharmingen): anti‐erythroid (TER‐119) and (optionally) anti‐CD19
  • Fluorescence activated cell sorter with 2‐laser, 4‐color capacity.
  • Additional reagents and equipment for mouse euthanasia (unit 1.8), cell counting ( appendix 3B), depletion of lineage‐positive cells by MACS (see protocol 1, steps to ), and flow cytometry (Chapter 5).

Basic Protocol 3: Isolation of Human Lymphohematopoietic Progenitors

  Materials
  • Human cord blood or bone marrow
  • MACS buffer (see recipe)
  • Direct CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec) including:
    • FcR Blocking Reagent
    • CD34 Microbeads
  • Labeled antibodies (BD Pharmingen and Caltag): FITC‐CD34, PE‐anti‐CD38, PE‐anti‐lineage markers (CD13, CD14, CD33 and CD64 for myeloid, glycophorin A for erythroid, CD19 for B lymphoid, CD3 and CD8 for T lymphoid, CD56 for NK cells) CD7, APC anti‐CD10
  • MACS positive‐selection (LS) columns (Miltenyi Biotec)
  • Midi MACS separation magnet and magnet stand (Miltenyi Biotec)
  • 15‐ml conical polypropylene tubes
  • Additional reagents and equipment required for Ficoll/Hypaque centrifugation (unit 7.1), cell counting ( appendix 3B), and cell sorting (Chapter 5)
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Figures

Videos

Literature Cited

Literature Cited
   Galy, A., Travis, M., Cen, D., and Chen, B. 1995. Human T, B, natural killer, and dendritic cells arise from a common bone marrow progenitor cell subset. Immunity 3:459‐473.
   Hao, Q.L., Zhu, J., Price, M.A., Payne, K.J., Barsky, L.W., and Crooks, G.M. 2001. Identification of a novel, human multilymphoid progenitor in cord blood. Blood 97:3683‐3690.
   Hardy, R.R., Carmack, C.E., Shinton, S.A., Kemp, J.D., and Hayakawa, K. 1991. Resolution and characterization of pro‐B and pre‐pro‐B cell stages in normal mouse bone marrow. J. Exp. Med. 173:1213‐1225.
   Igarashi, H., Gregory, S.C., Yokota, T., Sakaguchi, N., and Kincade, P.W. 2002. Transcription from the RAG1 locus marks the earliest lymphocyte progenitors in bone marrow. Immunity 17:117‐130.
   Kondo, M., Weissman, I.L., and Akashi, K. 1997. Identification of clonogenic common lymphoid progenitors in mouse bone marrow. Cell 91:661‐672.
   Kouro, T., Medina, K.L., Oritani, K., and Kincade, P.W. 2001. Characteristics of early murine B‐lymphocyte precursors and their direct sensitivity to negative regulators. Blood 97:2708‐2715.
   Rossi, M.I., Yokota, T., Medina, K.L., Garrett, K.P., Comp, P.C., Schipul, A.H. Jr., and Kincade, P.W. 2003. B lymphopoiesis is active throughout human life, but there are developmental age‐related changes. Blood 101:576‐584.
   Tudor, K.S., Payne, K.J., Yamashita, Y., and Kincade, P.W. 2000. Functional assessment of precursors from murine bone marrow suggests a sequence of early B lineage differentiation events. Immunity 12:335‐345.
   Yokota, T., Kouro, T., Hirose, J., Igarashi, H., Garrett, K.P., Gregory, S.C., Sakaguchi, N., Owen, J.J., and Kincade, P.W. 2003. Unique properties of fetal lymphoid progenitors identified according to RAG1 gene expression. Immunity 19:365‐375.
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