MagCellect Human CD4+ T Cell Isolation Kit
R&D Systems, part of Bio-Techne | Catalog # MAGH102
Key Product Details
Summary for MagCellect Human CD4+ T Cell Isolation Kit
Kit Summary
Why Isolate Regulatory T cells?
CD4+ T cells develop in the thymus and differentiate into various subsets of more specialized T lymphocytes. The development of these subsets is induced by distinct signals and is controlled by distinct transcription factors. CD4+ T cell subsets produce characteristic combinations of cytokines which enables them to exert diverse functions. CD4+ T cells are critically involved in recruiting and activating other immune cells, dampening ongoing immune responses, and maintaining immunologic memory. The relative expansion of CD4+ subsets changes dramatically in response to local environmental stresses or disease status. The isolation of CD4+ T cells is an important step in the preparation of multiple subsets including Th1, Th2, and Th17 cells. See Details
Prominent Characteristics of CD4+ T Cell Subsets
Th1 Cells
- Protect against intracellular infections
- Contribute to disease pathogenesis in autoimmunity
- Develop in response to IL-12 and type 1 Interferons
- Require transcription factors STAT4 and T-bet for differentiation and function
- Secrete IFN-gamma, TNF-alpha, IL-2
Th2 Cells
- Protect against intestinal helminths and extracellular bacteria
- Support B cell-dependent humoral immune responses
- Contribute to the development of allergic inflammation
- Develop in response to IL-4 and IL-2
- Require transcription factors STAT6 and GATA3 for differentiation and function
- Secrete IL-4, IL-5, IL-9, IL-13, IL-17E/IL-25
Th17 Cells
- Protect against extracellular bacteria and fungi
- Oppose some functions of regulatory T cells
- Mediate autoimmune and inflammatory disease pathogenesis
- Develop in response to TGF-beta and IL-6
- Expand and survive in the presence of IL-21 and IL-23
- Require transcription factor RORgammaT
- Secrete TNF-alpha, IL-6, IL-9, IL-17A, IL-17F, IL-21, IL-22, IL-26
Regulatory T cells (Treg)
- Limit the development and progression of immune responses
- Oppose some functions of Th17 cells
- Suppress the development of autoimmunity
- Develop in response to TGF-beta and IL-2
- Require transcription factor FoxP3 for differentiation and function
- Secrete TGF-beta, IL-9, IL-10, IL-35
Memory T cells (either CD4+ or CD8+)
- Survive for extended time after antigen is cleared
- Provide rapid immune activation in response to subsequent infection
- Differentiate into multiple subsets
- Reside in characteristic tissues depending on expressed profile of chemokine receptors
- Secrete IFN-gamma upon activation as well as other subset-specific cytokines
Other Subsets
Follicular helper T cells (Tfh) provide support for germinal center development and B cell responses. Their differentiation is under the control of the transcription factor Bcl6. Th9 cells and Th22 cells are characterized by the secretion of IL-9 and IL-22, respectively.
References:
Farber, D.L. et al. (2014) Nat. Rev. Immunol. 14:24.Magombedze, G. et al. (2013) Front. Physiol. 4:206.
Reagents Provided
The MagCellect Human CD4+ T Cell Isolation Kit contains the following reagents that allow for isolation of highly pure human CD4+ cells.
- MagCellect Human CD4+ T Cell Biotinylated Antibody Cocktail; 1 mL in PBS containing BSA
- MagCellect Streptavidin Ferrofluid; 1.25 mL in PBS containing BSA and preservatives
- MagCellect Plus Buffer (10X) – 10 mL of 10X concentrated buffer
*This kit contains sufficient reagents to process 1 x 109 total cells.
Stability and Storage
Store all reagents at 2 °C to 8 °C. DO NOT FREEZE.
CD4 is a transmembrane glycoprotein that is expressed predominantly on thymocytes and a subset of mature T lymphocytes. It is a standard phenotype marker for the identification of T cell populations. CD4 is expressed along with CD8 on double positive T cells during their development in the thymus. Either CD4 or CD8 expression is then lost, giving rise to single positive (SP) CD4+ or CD8+ mature T cells. CD4+ SP cells, also known as T helper cells, further differentiate into multiple subsets of CD4+ cells including Th1, Th2, Th9, Th17,Th22, Tfh, and Treg cells which regulate humoral and cellular immunity. In human, CD4 is additionally expressed on macrophages, neutrophils, monocytes, NK cells, and neurons and glial cells in the brain. CD4 binds directly to MHC class II molecules on antigen presenting cells. This interaction contributes to the formation of the immunological synapse which is focused around the TCR-MHC class II-antigenic peptide interaction. CD4 also functions as a chemotactic receptor for IL-16 and, in human, as a coreceptor for the gp120 surface glycoprotein of HIV-1.
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Enrichment of CD4+ T Cells from Human Peripheral Blood Mononuclear Cells Using the MagCellect CD4+ T Cell Isolation Kit. Ficolled human PBMC before (A) and after (B) isolation of CD4+ T cells 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). |
Assay Procedure
Refer to the product datasheet for complete product details.
Reagents Provided
Reagents Supplied in the MagCellect Human CD4+ T Cell Isolation Kit (Catalog # MAGH102):
- MagCellect Human CD4+ T Cell Biotinylated Antibody Cocktail; 1 mL in PBS containing BSA
- MagCellect Streptavidin Ferrofluid; 1.25 mL in PBS containing BSA and preservatives
- MagCellect Plus Buffer (10X) – 10 mL of 10X concentrated buffer
This kit contains sufficient reagents to process up to 1 x 109 total cells.
Other Supplies Required
- MagCellect Magnet (Catalog # MAG997) or equivalent
- 12 x 75 mm (5 mL) or 17 x 100 mm (15 mL) polystyrene round bottom tubes
- Sterile Pasteur pipettes or transfer pipettes
NOTE: Reaction incubations must be carried out at 2 to 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.
Procedure Overview
R&D Systems Protocol for the Magnetic Isolation of Human CD4+ T Cells
- Enrich for mononuclear cells by using a density gradient or any other method that provides a single cell suspension.
- Wash the cells 2 times with excess PBS.
- Centrifuge the cells for 10 minutes at 200 x g.
- Decant the supernatant. If necessary, remove red blood cells using R&D Systems Human Erythrocyte Lysing Kit (Catalog # WL1000).
- Resuspend the cells in a small volume of cold 1X MagCellect Plus Buffer.
- Perform a cell count.
- Adjust the cell concentration to 1 x 108 cells/mL with cold 1X MagCellect Plus Buffer.
- Transfer 20 x 107 cells (2.0 mL) into a 5 mL polystyrene tube.
- Add 200 μL of MagCellect Human CD4+ T Cell Biotinylated Antibody Cocktail.
- Mix the cell-antibody suspension and incubate at 2 °C to 8 °C for 15 minutes.
- Add 250 μL of MagCellect Streptavidin Ferrofluid to the cell suspension.
- Mix gently.
- Incubate at 2 °C to 8 °C for 15 minutes.
- Add 0.55 mL of cold 1X MagCellect Plus Buffer.
- Mix gently.
- Place the reaction tube in the MagCellect Magnet.
- Incubate for 6 minutes at room temperature (18 °C to 25 °C).
- Transfer the supernatant containing the CD4+ T cells into a new 5 mL tube.
- Repeat the magnetic depletion with the new tube containing the recovered cells. The supernatant obtained at the end of this step is the final depleted cell fraction containing the desired enriched CD4+ T cells.
Technical Hints
- If sterile cells are required following the cell selection, the entire procedure should be carried out in a laminar flow hood to maintain sterile conditions. Use sterile equipment when pipetting reagents that will be reused at a later date.
- Avoid antibody capping on cell surfaces and non-specific cell tagging by working fast, keeping cells and solutions cold through the use of pre-cooled solutions and by adhering to the incubation times and temperatures specified in the protocol. Increased temperature and prolonged incubation times may lead to non-specific cell labeling thus lowering cell purity and yield.
- When processing different numbers of cells observe the following guidelines: keep antibody cocktail and ferrofluid incubation times and temperatures the same; keep the cell density at 10 x 107 cells/mL; add 10 μL of the antibody cocktail per 1 x 107 cells being processed; add 12.5 μL of Streptavidin Ferrofluid per 1 x 107 cells being processed.
- When processing 20 x 107 cells or fewer, use the 12 x 75 mm (5 mL) tubes with the MagCellect Magnet horizontally positioned to accommodate up to six 5 mL tubes. Do not process more than 20 x 107 cells in each 5 mL tube and do not exceed a total reaction volume of 3 mL in each tube. A reaction volume of 2 mL is recommended for processing 10 x 107 cells. A reaction volume of 1 mL is recommended when processing 5 x 107 or fewer cells. Reaction volume adjustments must be made using 1X MagCellect Buffer just prior to the magnetic separation step.
- When processing greater than 20 x 107 cells, use the 17 x 100 mm (15 mL) tubes with the MagCellect magnet vertically positioned to accommodate up to two 15 mL tubes. Do not process more than 60 x 107 cells in each 15 mL tube and do not exceed a total reaction volume of 9 mL in each tube. When using this larger tube, increase the reaction volume before the magnetic separation step according to the following formula: 3 mL for each 20 x 107 cells processed. Also increase the magnetic incubation time described in to 8 minutes. Reaction volume adjustments must be made using 1X MagCellect Buffer just prior to the magnetic separation step.