MagCellect Mouse Naive CD4+ T Cell Isolation Kit Summary
For the isolation of mouse naïve CD4+ T cells.
Why Isolate Naïve CD4+ T cells?
Mouse CD4+ T cells develop in the thymus and are released as naïve T cells that also express CD62L and low levels of CD44. 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.
The MagCellect Mouse Naïve CD4+ T Cell Isolation Kit contains the following reagents that allow for isolation of highly pure mouse naïve CD4+ T cells.
MagCellect Mouse Naïve CD4+ T Cell Biotinylated Antibody Cocktail
MagCellect Streptavidin Ferrofluid
MagCellect Plus Buffer (10X)
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.
MagCellect Mouse Naive CD4+ T Cell Isolation Kit Scientific Data Examples
Enrichment of Naïve CD4+ T Cells from Mouse Splenocytes.
Ficolled mouse splenocytes before (A) and after (B) processing with the MagCellect Mouse Naïve CD4+ T Cell Isolation Kit. Cells were stained with FITC-conjugated Rat Anti-Mouse CD44 Monoclonal Antibody and PE-conjugated Rat Anti-Mouse CD62L Monoclonal Antibody (Catalog # FAB5761P).
Preparation & Storage
The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Store the unopened product at 2 - 8 °C. Do not use past expiration date.
MagCellect Magnet (Catalog # MAG997) or equivalent
12 x 75 mm (5 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.
R&D Systems Protocol for the Magnetic Isolation of Mouse Regulatory T Cells
Prepare a single cell suspension of mononuclear cells.
Wash the cells once 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 Mouse Erythrocyte Lysing Kit (Catalog # WL2000).
Resuspend the cells in a small volume of cold 1X MagCellect Plus Buffer.
Perform a cell count.
Adjust the cell concentration to at least 20 x 107 cells/mL with cold 1X MagCellect Plus Buffer.
Transfer 20 x 107 cells (1 mL) into a 5 mL polystyrene tube.
Add 200 μL of MagCellect Mouse Naïve 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.
Incubate at 2 °C to 8 °C for 15 minutes.
Add 1.55 mL of MagCellect Plus Buffer.
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 naïve 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 naïve CD4+ T cells.
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 20 x 107 cells/mL; add 10 μL of the antibody cocktail per 1 x 107 cells being processed; add 10 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.
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