This protocol provides a basic guide for the magnetic selection of CD4+ T lymphocytes from preparations of peripheral blood mononuclear cells (PBMC) or splenocytes.
In this protocol, a mononuclear cell suspension is first incubated with a biotinylated antibody cocktail which targets unwanted cells. Streptavidin ferrofluid is added to the reaction, and the streptavidin coated nanoparticles interact with the biotinylated antibody tagged cells. The tube containing the cell suspension is then placed within a magnetic field. Magnetically tagged cells (unwanted cell fraction) will migrate toward the magnet, leaving the untagged cells (desired cell population) in suspension to be harvested by aspiration while the tube remains in the magnetic field. The enriched cell preparation is then available for a variety of applications including tissue culture, immune status monitoring, and flow cytometry.
CD4+ T cells develop in the thymus and differentiate into various subsets of more specialized T lymphocytes. These cells differentiate into subsets of specialized effector T lymphocytes in response to distinct environmental cues and the activation of specific transcription factors. The isolation of CD4+ T cells is an important step in the preparation of multiple subsets including Th1, Th2, Th17, Th9, Th22, and regulatory T cells. CD4+ T cell subsets secrete characteristic combinations of cytokines which enables them to exert diverse functions including the recruitment and activation of additional immune cells, the dampening of ongoing immune responses, and the maintenance of immunologic memory.
PBMC and splenocyte suspensions are suitable starting samples for the isolation of CD4+ T cells. Follow the instructions in this protocol to obtain PBMC and splenocytes.
MagCellect™ CD4+ T cell isolation kits are designed to isolate CD4+ T cells by negative selection. The resulting cell preparations are highly enriched with CD4+ T cells with no detectable CD8+ T cells. MagCellect kits are available for the isolation of CD4+ T cells (human or mouse), naïve CD4+ T cells (human or mouse), and memory CD4+ T cells (human).These kits contain sufficient quantities of the following reagents to process 1 x 109 total cells:
This procedure is for processing 2 x 108 total cells using 5 mL tubes and the MagCellect Magnet. For processing other quantities of cells please refer to the Technical Hints section of this protocol. Cells and reagents should be kept cold using an ice bath or a refrigerator. Reaction incubations must be carried out at 2-8 °C in a refrigerator and not in an ice bath to avoid excessively low temperatures that can slow the kinetics of the optimized reactions.
View Larger Image |
Enrichment of CD4+ T Cells from Peripheral Blood Mononuclear Cells Using the MagCellect Human CD4+ T Cell Isolation Kit. Ficolled human PBMC before (A) and after (B) isolation of CD4+ T cells using the MagCellect Human CD4+ T Cell Isolation Kit (Catalog # MAGH102) were stained with PE-conjugated Mouse Anti-Human CD3 epsilon Monoclonal Antibody (Catalog # FAB100P) and FITC-conjugated Mouse Anti-Human CD4 Monoclonal Antibody (Catalog # FAB3791F). |
View Larger Image |
Enrichment of Naïve CD4+ T Cells from PBMC Using the MagCellect Human Naïve CD4+ T Cell Isolation Kit. Ficolled human PBMCs before (A) and after (B) processing with the MagCellect Human Naïve CD4+ T Cell Isolation Kit (Catalog # MAGH115). Cells were stained with an APC-conjugated Mouse Anti-Human CD4 Monoclonal Antibody (Catalog # FAB3791A) and PE-conjugated Mouse Anti-Human CD45RA Monoclonal Antibody (Catalog # FAB1430P). |
Leukocyte Preparation Protocol
Troubleshooting Guide for MagCellect Cell Isolation Kits
MagCellect Cell Selection Kits & Reagents
Flow Cytometry Protocol for Staining Membrane-associated Proteins in Suspended Cells
Flow Cytometry Protocol for Analysis of Cell Viability using Propidium Iodide
Flow Cytometry Protocol for Analysis of Cell Viability using 7-Amino Actinomycin D (7-AAD)