Mouse CD4+ T Cell Enrichment Column, Small, Pack of 4
Mouse CD4+ T Cell Enrichment Column, Small, Pack of 4 Summary
For the isolation of mouse CD4+ T cells.
- Yields a highly pure (84-91%) population of CD4+ T cells
- Separates cells based on gravitational flow
- Negative selection reduces sources of experimental variation
Why Isolate CD4+ T cells?
CD4+ T cells develop in the thymus and 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 ongoing immune responses, and the maintenance of immunologic memory.
The Mouse CD4+ T Cell Enrichment Column Kit, Small contains the following components to isolate mouse CD4+ T cells.
- Mouse CD4+ T Cell Subset Columns
- Monoclonal Antibody Cocktail
- Column Buffer Concentrate (10X)
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.
Refer to the product datasheet for complete product details.
Briefly, mouse CD4+ T cells can be isolated using the following procedure:
- Incubate a single-cell suspension of leukocytes with the Monoclonal Antibody Cocktail
- Apply antibody-treated cell suspension to the CD4+ T Cell Subset Column
- Elute CD4+ T cells with 1X Column Buffer
The Mouse CD4+ T Cell Enrichment Column Kit, Small (Catalog #MCD4C-1000) contains the following components to allow for the isolation of mouse CD4+ T cells.
- Mouse CD4+ T Cell Subset Columns
- Monoclonal Antibody Cocktail
- Column Buffer Concentrate (10X)
Other Supplies Required
- In order to best determine column performance, we advise that users retain a small portion of the starting cell population. Following cell selection with the column, it is then possible to perform immunophenotyping analysis on both starting and eluted cells. This information, when combined with the actual number of cells loaded and recovered, can then be used to calculate the percentage recovery of the target cell population.
- Care should be taken during cell preparation to reduce the number of platelets before loading the cells onto the column. Platelet contamination can be reduced by performing a final wash centrifugation of the cells at a reduced speed (150 - 200 x g).
- Some of the salts in the 10X column buffer solution may precipitate after storage at 4 °C. Should this be the case, do not carry out the 1:10 buffer dilution until all salts are in solution. This may be achieved by warming the 10X column buffer bottle in a 37 °C water bath for 5 - 10 minutes. Once there is no longer evidence of precipitates, the 10X column buffer may be diluted 1:10 to prepare the 1X column buffer for column processing.
Helpful Hints in Running T Cell Enrichment Columns
- Remove as many clumps as possible from the cell suspension before loading cells onto the column. Although the column is designed to filter out larger clumps of cells, too many clumps on the filter will reduce the column flow rate and cell recovery. In addition, leaving a large number of cells in a small volume of buffer for more than 30 minutes may promote cell clumping.
- If cells do not move into column after 15 minutes, the filter may have become clogged. If this occurs, move the white filter at the top of the column to the side with a sterile pipette. The cells should migrate into the column more easily.
- The column is designed so that the white filter at the top of the column bed will stop buffer flow and prevent the column from drying out. However leaving the open column exposed to air for more than 1 hour may cause the column bed to dry out and the CD4+ T cell enrichment to be compromised.
- Cell recovery after column processing is largely dependent on the total number of cells loaded. Optimal column performance is achieved by loading approximately 75 x106 cells. Loading fewer cells will negatively affect total cell recovery.
- If buffer does not drip out of column after initial removal of the bottom cap, try tapping the side of the column to remove any air locks that may be preventing the flow of buffer.
- The white filter at the top of the column bed may be found floating as a result of being dislodged during shipping. The column can still be used for cell processing after pushing the white filter onto the column bed with the back end of a sterile 2 mL pipette. The white filter should be positioned close to the column bed.
R&D Systems Protocol for Mouse CD4+ T Cell Enrichment Column, Small
Prepare a single cell suspension of mononuclear cells.
Wash the cells with excess sterile PBS.
Remove the red blood cells if necessary (e.g., using R&D Systems Mouse Erythrocyte Lysing Kit Catalog # WL2000)
Resuspend the cells in a small volume of 1X Column Wash Buffer.
Perform a cell count.
Adjust the cell concentration to 1-2 x 108 cells/mL with 1X Column Wash Buffer.
Mix 2 x 108 suspended cells with the contents of one vial of Monoclonal Antibody Cocktail.
Incubate for 15 minutes at room temperature.
Wash cells twice with 10 mL 1X Column Wash Buffer.
Resuspend cells in 2 mL of 1X Column Wash Buffer.
Load cell suspension onto the prepared column.
Incubate for 10 minutes at room temperature.
Elute CD4+ T cells with 10 mL of 1X Column Wash Buffer.
Collect the cells in a sterile 50 mL centrifuge tube.
Pellet the cells using centrifugation.
Resuspend CD4+ T cells in the appropriate buffer or culture medium for downstream applications.
Citations for Mouse CD4+ T Cell Enrichment Column, Small, Pack of 4
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.
Citations: Showing 1 - 10
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PD-1+ melanocortin receptor dependent-Treg cells prevent autoimmune disease
Authors: F Muhammad, D Wang, A Montieth, S Lee, J Preble, CS Foster, TA Larson, K Ding, JD Dvorak, DJ Lee
Sci Rep, 2019;9(1):16941. 2019
Symbionts exploit complex signaling to educate the immune system
Authors: D Erturk-Has, SF Oh, NA Okan, G Stefanetti, FS Gazzaniga, PH Seeberger, SE Plevy, DL Kasper
Proc. Natl. Acad. Sci. U.S.A., 2019;0(0):. 2019
Amiselimod (MT-1303), a novel sphingosine 1-phosphate receptor-1 functional antagonist, inhibits progress of chronic colitis induced by transfer of CD4+CD45RBhighï¿½T cells
Authors: K Shimano, Y Maeda, H Kataoka, M Murase, S Mochizuki, H Utsumi, K Oshita, K Sugahara
PLoS ONE, 2019;14(12):e0226154. 2019
High Thymic Output of Effector CD4+ Cells May Lead to a Treg?:?T Effector Imbalance in the Periphery in NOD Mice
Authors: Y Zhao, P Alard, MM Kosiewicz
J Immunol Res, 2019;2019(0):8785263. 2019
?-Galactosylceramide treatment before allergen sensitization promotes iNKT cell-mediated induction of Treg cells, preventing Th2 cell responses in murine asthma
Authors: Q Chen, X Guo, N Deng, L Liu, S Chen, A Wang, R Li, Y Huang, X Ding, H Yu, S Hu, H Nie
J. Biol. Chem., 2019;0(0):. 2019
Cannabidiol (CBD) induces functional Tregs in response to low-level T cell activation
Authors: Barbara L F Kaplan
Cell. Immunol., 2016;0(0):. 2016
Regulatory T Cells Control Th2-Dominant Murine Autoimmune Gastritis
Authors: Jessica Harakal
J Immunol, 2016;197(1):27-41. 2016
Defect density in multiwalled carbon nanotubes influences ovalbumin adsorption and promotes macrophage activation and CD4(+) T-cell proliferation
Int J Nanomedicine, 2016;11(0):4357-71. 2016
Digoxin Inhibits Induction of Experimental Autoimmune Uveitis in Mice, but Causes Severe Retinal Degeneration
Invest. Ophthalmol. Vis. Sci., 2016;57(3):1441-7. 2016
Both rejection and tolerance of allografts can occur in the absence of secondary lymphoid tissues.
Authors: Kant C, Akiyama Y, Tanaka K, Shea S, Yamada Y, Connolly S, Marino J, Tocco G, Benichou G
J Immunol, 2015;194(3):1364-71. 2015
Dynamic motile T cells highly respond to the T cell stimulation via PI3K-Akt and NF-kappaB pathways.
Authors: Kim H, Na B, Kwon M, Ko Y, Han W, Jun C
PLoS ONE, 2013;8(3):e59793. 2013
Increased membrane cholesterol in lymphocytes diverts T-cells toward an inflammatory response.
Authors: Surls J, Nazarov-Stoica C, Kehl M, Olsen C, Casares S, Brumeanu TD
PLoS ONE, 2012;7(6):e38733. 2012
Antigen-specific Th9 cells exhibit uniqueness in their kinetics of cytokine production and short retention at the inflammatory site.
Authors: Tan C, Aziz MK, Lovaas JD, Vistica BP, Shi G, Wawrousek EF, Gery I
J. Immunol., 2010;185(11):6795-801. 2010
NKT cells mediate pulmonary inflammation and dysfunction in murine sickle cell disease through production of IFN-gamma and CXCR3 chemokines.
Authors: Wallace KL, Marshall MA, Ramos SI, Lannigan JA, Field JJ, Strieter RM, Linden J
Blood, 2009;114(3):667-76. 2009
T-bet inhibits both TH2 cell-mediated eosinophil recruitment and TH17 cell-mediated neutrophil recruitment into the airways.
Authors: Fujiwara M, Hirose K, Kagami S, Takatori H, Wakashin H, Tamachi T, Watanabe N, Saito Y, Iwamoto I, Nakajima H
J. Allergy Clin. Immunol., 2007;119(3):662-70. 2007
TLR4 mediates vaccine-induced protective cellular immunity to Bordetella pertussis: role of IL-17-producing T cells.
Authors: Higgins SC, Jarnicki AG, Lavelle EC, Mills KH
J. Immunol., 2006;177(11):7980-9. 2006
Leptin neutralization interferes with pathogenic T cell autoreactivity in autoimmune encephalomyelitis.
Authors: De Rosa V, Procaccini C, La Cava A, Chieffi P, Nicoletti GF, Fontana S, Zappacosta S, Matarese G
J. Clin. Invest., 2006;116(2):447-55. 2006
Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3.
Authors: Chen, WanJun, Jin, Wenwen, Hardegen, Neil, Lei, Ke-Jian, Li, Li, Marinos, Nancy, McGrady, George, Wahl, Sharon M
J Exp Med, 2003;198(12):1875-86. 2003
CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production.
Authors: Thornton, A M, Shevach, E M
J Exp Med, 1998;188(2):287-96. 1998
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Have been using this kit in our laboratory for over 6 years in order to isolate CD4+ T cells for in vitro assays. Tim and time again these columns have yielded 90% or greater CD4+ T cell purity. I would recommend keeping lymph node and splenic cells separate when using the columns as they have a tendency to clump when put together before running through the columns.