MagCellect Mouse CD4+ T Cell Isolation Kit Summary
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Enrichment of CD4+ T Cells from Mouse Splenocytes Using the MagCellect CD4+ T Cell Isolation Kit. Ficolled mouse splenocytes before (A) and after (B) isolation of CD4+ T cells were stained with PE-conjugated Rat Anti-Mouse CD4 Monoclonal Antibody (Catalog # FAB554P) and FITC-conjugated Rat Anti-Mouse CD3 Monoclonal Antibody (Catalog # FAB4841F).
Background: CD4
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.
Assay Procedure
Refer to the product datasheet for complete product details.
Briefly, mouse CD4+ T cells can be isolated using the following procedure:
- Incubate the single-cell suspension of splenocytes with the MagCellect Antibody Cocktail
- Add the MagCellect Streptavidin Ferrofluid
- Place the tube in a magnetic field
- Collect the desired cells while undesired cells remain attracted to the magnet
Reagents Supplied in the MagCellect Mouse CD4+ T Cell Isolation Kit (Catalog # MAGM202):
- MagCellect Mouse 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.
- 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.
R&D Systems Protocol for the Magnetic Isolation of Mouse CD4+ T Cells
Prepare a single cell suspension of splenocytes.
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 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 20 x 107 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 Mouse 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 1.6 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.
Citations for MagCellect Mouse CD4+ T Cell Isolation Kit
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
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Citations: Showing 1 - 10
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Inhibition of MicroRNA-23b Attenuates Immunosuppression in Late Sepsis through NIK, TRAF1 and XIAP
Authors: H Zhang, H Li, A Shaikh, Y Caudle, B Yao, D Yin
J. Infect. Dis., 2018-06-20;0(0):. 2018-06-20
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Gastric LTi cells promote lymphoid follicle formation but are limited by IRAK-M and do not alter microbial growth.
Authors: Shiu J, Piazuelo M, Ding H, Czinn S, Drakes M, Banerjee A, Basappa N, Kobayashi K, Fricke W, Blanchard T
Mucosal Immunol, 2015-01-21;8(5):1047-59. 2015-01-21
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Apoptotic programs are determined during lineage commitment of CD4+ T effectors: selective regulation of T effector-memory apoptosis by inducible nitric oxide synthase.
Authors: Purushothaman D, Marcel N, Garg M, Venkataraman R, Sarin A
J Immunol, 2012-12-05;190(1):97-105. 2012-12-05
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Loss of AMPK exacerbates experimental autoimmune encephalomyelitis disease severity.
Authors: Nath N, Khan M, Rattan R, Mangalam A, Makkar RS, de Meester C, Bertrand L, Singh I, Chen Y, Viollet B, Giri S
Biochem. Biophys. Res. Commun., 2009-05-30;386(1):16-20. 2009-05-30
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A microbial symbiosis factor prevents intestinal inflammatory disease.
Authors: Mazmanian SK, Round JL, Kasper DL
Nature, 2008-05-29;453(7195):620-5. 2008-05-29
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B cell mediated priming following pneumococcal colonization.
Authors: Rabquer B, Shriner AK, Smithson SL, Westerink MA
Vaccine, 2006-11-28;25(0):2036. 2006-11-28
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Evidence for a role for notch signaling in the cytokine-dependent survival of activated T cells.
Authors: Bheeshmachar G, Purushotaman D, Sade H, Gunasekharan V, Rangarajan A, Sarin A
J. Immunol., 2006-10-15;177(8):5041-50. 2006-10-15
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Soluble factors from Leishmania major-specific CD4+T cells and B cells limit L. amazonensis amastigote survival within infected macrophages.
Authors: Mukbel R, Petersen CA, Jones DE
Microbes Infect., 2006-08-01;8(9):2547-55. 2006-08-01
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T-bet binding to newly identified target gene promoters is cell type-independent but results in variable context-dependent functional effects.
Authors: Beima KM, Miazgowicz MM, Lewis MD, Yan PS, Huang TH, Weinmann AS
J. Biol. Chem., 2006-02-10;281(17):11992-2000. 2006-02-10
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CD4+ T-cell reconstitution reduces cytomegalovirus in the immunocompromised brain.
Authors: Reuter JD, Wilson JH, Idoko KE, van den Pol AN
J. Virol., 2005-08-01;79(15):9527-39. 2005-08-01
FAQs
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When using MacCellect Cell Isolation kits (MAGM*** or MAGH***), why is it important to lyse red blood cells before starting the cell isolation protocol?
It is important to remove red blood cells before the isolation protocol using MagCellect isolation kits because not doing so can cause a poorer yield of selected cells. Red blood cells become sticky during the incubation protocols and may interfere with the interactions needed for removal of unwanted populations.
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