MagCellect Mouse Naive CD4+ T Cell Isolation Kit

Catalog # Availability Size / Price Qty
MAGM205
Enrichment of Naïve CD4+ T Cells from Mouse Splenocytes.
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MagCellect Mouse Naive CD4+ T Cell Isolation Kit Summary

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.

 
Reagents Provided

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.

Specifications

Shipping Conditions
The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.
Species
Mouse

Product Datasheets

Data Example

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).

Background: CD4

Entrez Gene IDs
920 (Human); 12504 (Mouse); 24932 (Rat); 403931 (Canine); 101864991 (Cynomolgus Monkey); 493775 (Feline)
Alternate Names
CD_antigen: CD4; CD4 antigen (p55); CD4 antigen; CD4 molecule; CD4 receptor; CD4; CD4mut; T-cell surface antigen T4/Leu-3; T-cell surface glycoprotein CD4

Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, mouse naïve CD4+ T cells can be isolated using the following procedure:

  • Incubate the single-cell suspension 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
 

 

Kit Components

The MagCellect Mouse CD4+ T Cell Isolation Kit (Catalog # MAGM205) contains the following reagents:

  • MagCellect Mouse CD4+ T Cell Biotinylated Antibody Cocktail
  • MagCellect Streptavidin Ferrofluid
  • MagCellect Plus Buffer (10X)

This kit contains sufficient reagents to process up to 1 x 109 total cells.

 

 

Other Supplies Required

Reagents

  • Ficoll-Hypaque™
  • Hemocytometer
  • Centrifuge
  • Mouse Erythrocyte Lysing Kit (Catalog # WL2000)
  • 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.

 

 

Procedure Overview

R&D Systems Protocol for the Magnetic Isolation of Mouse Regulatory T Cells

PBMC Preparation

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.

Prepare a single cell suspension of mononuclear cells.

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.

Decant the supernatant.

Perform a cell count.

Adjust the cell concentration to at least 20 x 107 cells/mL with cold 1X MagCellect Plus Buffer.

Perform a cell count.

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.

Protocol for Human Monocyte-derived Dendritic Cell Differentiation Step 4

Add 250 μL of MagCellect Streptavidin Ferrofluid to the cell suspension.

Mix gently.

Incubate at 2 °C to 8 °C for 15 minutes.

Add 250 μL of MagCellect Streptavidin Ferrofluid to the cell suspension.

Add 1.55 mL of MagCellect Plus Buffer.

Mix gently.

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.

Place the reaction tube in the MagCellect Magnet.

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.

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.
 

 

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 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.

Citations for MagCellect Mouse Naive 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.

13 Citations: Showing 1 - 10
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  1. CCL11 increases the proportion of CD4+CD25+Foxp3+ Treg cells and the production of IL?2 and TGF?&beta by CD4+ T�cells via the STAT5 signaling pathway
    Authors: R Wang, K Huang
    Mol Med Rep, 2020;0(0):.  2020
  2. Critical role of Tim-3 mediated autophagy in chronic stress induced immunosuppression
    Authors: A Qin, T Zhong, H Zou, X Wan, B Yao, X Zheng, D Yin
    Cell Biosci, 2019;9(0):13.  2019
  3. Enhanced susceptibility to chemically induced colitis caused by excessive endosomal TLR signaling in LRBA-deficient mice
    Authors: KW Wang, X Zhan, W McAlpine, Z Zhang, JH Choi, H Shi, T Misawa, T Yue, D Zhang, Y Wang, S Ludwig, J Russell, M Tang, X Li, AR Murray, EMY Moresco, EE Turer, B Beutler
    Proc. Natl. Acad. Sci. U.S.A., 2019;116(23):11380-11389.  2019
  4. Environmental cues received during development shape dendritic cell responses later in life
    Authors: JL Meyers, B Winans, E Kelsaw, A Murthy, S Gerber, BP Lawrence
    PLoS ONE, 2018;13(11):e0207007.  2018
  5. Integrated STAT3 and Ikaros Zinc Finger Transcription Factor Activities Regulate Bcl-6 Expression in CD4(+) Th Cells
    Authors: KA Read, MD Powell, CE Baker, BK Sreekumar, VM Ringel-Sca, H Bachus, RE Martin, ID Cooley, IC Allen, A Ballestero, KJ Oestreich
    J. Immunol., 2017;0(0):.  2017
  6. Basophils as a primary inducer of the T helper type 2 immunity in ovalbumin-induced allergic airway inflammation.
    Authors: Zhong W, Su W, Zhang Y, Liu Q, Wu J, Di C, Zhang Z, Xia Z
    Immunology, 2014;142(2):202-15.  2014
  7. Attenuation of experimental colitis in glutathione peroxidase 1 and catalase double knockout mice through enhancing regulatory T cell function.
    Authors: Kim H, Lee A, Choi E, Kie J, Lim W, Lee H, Moon B, Seoh J
    PLoS ONE, 2014;9(4):e95332.  2014
  8. TNF-like ligand 1A (TL1A) gene knockout leads to ameliorated collagen-induced arthritis in mice: implication of TL1A in humoral immune responses.
    Authors: Wang X, Hu Y, Charpentier T, Lamarre A, Qi S, Wu J, Luo H
    J Immunol, 2013;191(11):5420-9.  2013
  9. Modeling and experimental methods to probe the link between global transcription and spatial organization of chromosomes.
    Authors: Iyer K, Maharana S, Gupta S, Libchaber A, Tlusty T, Shivashankar G
    PLoS ONE, 2012;7(10):e46628.  2012
  10. Developmental heterogeneity in DNA packaging patterns influences T-cell activation and transmigration.
    Authors: Gupta S, Marcel N, Talwar S, Garg M, R I, Perumalsamy L, Sarin A, Shivashankar G
    PLoS ONE, 2012;7(9):e43718.  2012
  11. The effect of conditional EFNB1 deletion in the T cell compartment on T cell development and function.
    Authors: Jin W, Qi S, Luo H
    BMC Immunol., 2011;12(0):68.  2011
  12. Tolerogenic signals delivered by dendritic cells to T cells through a galectin-1-driven immunoregulatory circuit involving interleukin 27 and interleukin 10.
    Authors: Ilarregui JM, Croci DO, Bianco GA, Toscano MA, Salatino M, Vermeulen ME, Geffner JR, Rabinovich GA
    Nat. Immunol., 2009;10(9):981-91.  2009
  13. Differential glycosylation of TH1, TH2 and TH-17 effector cells selectively regulates susceptibility to cell death.
    Authors: Toscano MA, Bianco GA, Ilarregui JM, Croci DO, Correale J, Hernandez JD, Zwirner NW, Poirier F, Riley EM, Baum LG, Rabinovich GA
    Nat. Immunol., 2007;8(8):825-34.  2007

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