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Isolation of Mouse Mononuclear Cells

Ada M. Kruisbeek1

1Netherlands Cancer Institute, Amsterdam, The Netherlands, Netherlands

Publication Name: 
Current Protocols in Immunology
Unit Number: 
UNIT 3.1
DOI: 
10.1002/0471142735.im0301s39
Online Posting Date: 
May, 2001
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Abstract

This unit covers techniques for preparing lymphoid cell suspensions and removing red blood cells and dead cells. These basic manipulations are required for most of the subsequent protocols in this chapter, i.e., measuring functional responses of mouse lymphocytes.

     
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Table of Contents

  • Section I: Isolation and Fractionation of Mononuclear Cell Populations
  • Unit Introduction
  • Basic Protocol: Preparation of Cell Suspensions from Spleen, Thymus, and Lymph Node
  • Support Protocol: Removal of Red Blood Cells from Spleen Cell Suspensions
  • Support Protocol: Removal of Dead Cells Using the One-Step Gradient Method
  • Reagents and Solutions
  • Commentary
  • Tables
     
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Materials

Basic Protocol: Preparation of Cell Suspensions from Spleen, Thymus, and Lymph Node

 Materials
  • Complete RPMI-5 or DMEM-5 media (appendix 2)
  • Freshly removed organs from mice: £6 weeks old for thymus; 6 weeks to 6 months old for spleen and lymph node (unit 1.9)
  • 60 × 15–mm petri dishes
  • Scissors and forceps, kept in sterile beaker with 70% ethanol
  • 6-ml syringe with 19-G needle
  • 200-µm mesh nylon screen
  • Sorvall H-1000B rotor (or equivalent)
  • Additional reagents and equipment for CO2 asphyxiation or cervical dislocation of mice (unit 1.8) and lymphoid organ removal (unit 1.9)

Support Protocol: Removal of Red Blood Cells from Spleen Cell Suspensions

 Additional Materials
  • ACK lysing buffer

Support Protocol: Removal of Dead Cells Using the One-Step Gradient Method

 Additional Materials
  • High-density solution: Ficoll-Paque (Ficoll and sodium diatrizoate; Pharmacia LKB) or Lympholyte-M (Cedarlane)
  • 12- or 50-ml polypropylene centrifuge tubes

NOTE: The densities of Ficoll-Paque and Lympholyte-M are temperature dependent; this protocol and the commercially available high-density solutions have been designed for use at room temperature. Care should therefore be taken to bring cell suspensions, centrifuge, and high-density solutions to room temperature. Failure to do so will result in loss of live cells from the interface—instead of floating to the top of the high-density layer, they will collect at the bottom of the tube.

Looking for materials?  Suppliers Guide
     
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