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23428
Human Activated T Cell Markers Flow Cytometry Panel
Flow Cytometry Kits & Reagents
Assay Kit

Human Activated T Cell Markers Flow Cytometry Panel #23428

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Flow Cytometry Image 1: Human Activated T Cell Markers Flow Cytometry Panel
Flow cytometric analysis of live human peripheral blood mononuclear cells, untreated (blue) or treated with TPA (12-O-Tetradecanoylphorbol-13-Acetate) #4174 (0.081 uM, 5 hr) and Ionomycin, Calcium Salt #9995 (1.34 uM, 5 hr; green), using CD69 (FN50) Mouse mAb (PE-Cy7®Conjugate) (solid lines) or concentration-matched Mouse (MOPC-21) mAb IgG1 Isotype Control (PE-Cy7® Conjugate) #79339 (dashed lines).
Flow Cytometry Image 2: Human Activated T Cell Markers Flow Cytometry Panel
Flow cytometric analysis of live human peripheral blood mononuclear cells using IL2-Rα/CD25 (BC96) Mouse mAb (APC Conjugate) (right) compared to concentration-matched Mouse (MOPC-21) mAb IgG1 Isotype Control (APC Conjugate) #49083 (left), co-stained with CD4 (RPA-T4) Mouse mAb (FITC Conjugate) #48705.
Flow Cytometry Image 3: Human Activated T Cell Markers Flow Cytometry Panel
Flow cytometric analysis of live human peripheral blood mononuclear cells using CD8α (RPA-T8) Mouse mAb (FITC Conjugate) (solid line) compared to concentration-matched Mouse (MOPC-21) mAb IgG1 Isotype Control (FITC Conjugate) #97146 (dashed line).
Flow Cytometry Image 4: Human Activated T Cell Markers Flow Cytometry Panel
Flow cytometric analysis of live human peripheral blood mononuclear cells using CD4 (RPA-T4) Mouse mAb (violetFluor 450 Conjugate) (solid line) compared to concentration-matched Mouse (MOPC-21) mAb IgG1 Isotype Control (violetFluor 450 Conjugate) #40282 (dashed line).
Flow Cytometry Image 5: Human Activated T Cell Markers Flow Cytometry Panel
Flow cytometric analysis of live human peripheral blood mononuclear cells using CD3 (UCHT1) Mouse mAb (PE Conjugate) (solid line) compared to concentration-matched Mouse (MOPC-21) mAb IgG1 Isotype Control (PE Conjugate) #63630 (dashed line).
To Purchase # 23428
Cat. # Size Qty. Price
23428S
1 Kit  (100 assays)
$ 940

Important Ordering Details

Custom Ordering Details: Product is assembled upon order. Please allow up to three business days for your product to be processed.

Protocol

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Flow Cytometry, Live Cell Protocol for Directly Conjugated Antibodies

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100 ml 10X PBS (#12528) to 900 ml water, mix.
  2. Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616), or prepare a 0.5% BSA PBS buffer by dissolving 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com for a full listing of cellular dyes validated for use in flow cytometry.

B. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to immunostaining.

NOTE: Human Fc receptors cross-react with rabbit IgG. When cells of interest express high levels of Fc receptor protein (for example, macrophage/monocyte lineages), pre-incubate live cells with human Fc block prior to immunostaining with rabbit antibodies.

NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.

  1. Aliquot desired number of cells into tubes or wells. (Generally, 5x105 to 1x106 cells per assay.)
  2. Pellet cells by centrifugation and remove supernatant.
  3. Resuspend cells in 100 µl of diluted primary antibody, prepared in Antibody Dilution Buffer at a recommended dilution or as determined via titration.
  4. Incubate for 30 min to 1 hr on ice. Protect from light.
  5. Wash by centrifugation in Antibody Dilution Buffer. Discard supernatant. Repeat.
  6. Resuspend cells in 200-500 µl of Antibody Dilution Buffer and analyze on flow cytometer.

posted June 2017

revised January 2022

Protocol Id: 1504

Product Description

The Human Activated T Cell Markers Flow Cytometry Panel can be used to identify activated human T cells.

T cells are identified by expression of CD3. There are two major subsets of conventional T cells: helper T cells which express CD4, and cytotoxic T cells which express CD8. Upon stimulation through the T cell receptor, T cells upregulate expression of CD69 and IL-2Rα/CD25. Regulatory T cells constitutively express IL-2Rα/CD25.

Product Usage Information

All antibodies in this kit are compatible with the Flow Cytometry, Live Cell Protocol for Directly Conjugated Antibodies and can be used in a single staining mix. After antibody staining and prior to acquisition on a flow cytometer, we recommend adding Propidium Iodide or 7-AAD to enable identification and exclusion of dead cells from the analysis.

Gating strategy for identifying activated T cells:
If Propidium Iodide or 7-AAD was used, first gate on viable cells. Next, gate on lymphocytes based on forward scatter and side scatter. Helper T cells are the CD3+CD4+ cells within the lymphocyte gate. Activated helper T cells are positive for expression of CD69 and/or IL-2Rα/CD25. Regulatory T cells can also express IL-2Rα/CD25. Cytotoxic T cells are the CD3+CD8+ cells within the lymphocyte gate. Activated cytotoxic T cells are positive for expression of CD69 and/or IL-2Rα/CD25.

Storage

Antibodies are supplied in 10 mM NaH2PO4, 150 mM NaCl, 0.09% NaN3, 0.1% gelatin, pH7.2. Store at 4oC. Do not aliquot the antibodies. Protect from light. Do not freeze.

All components in this kit are stable in accordance with the date printed on the outer packaging label when stored at the recommended temperature. Please refer to product labels, datasheets, or web pages for specific "Best By" dates for each individual component.

Specificity / Sensitivity

Each antibody in the Human Activated T Cell Markers Flow Cytometry Panel detects endogenous levels of its target protein. CD3 (UCHT1) Mouse mAb (PE Conjugate), CD4 (RPA-T4) Mouse mAb (violetFluor™ 450 Conjugate), CD8α (RPA-T8) Mouse mAb (FITC Conjugate), IL2-Rα/CD25 (BC96) Mouse mAb (APC Conjugate), and CD69 (FN50) Mouse mAb (PE-Cy7® Conjugate) detect epitopes within the extracellular domains.

Species Reactivity:

Human

Source / Purification

Monoclonal antibodies were purified from tissue culture supernatant via affinity chromatography. The purified antibodies were conjugated under optimal conditions, with unreacted dye removed from the preparation.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

For Research Use Only. Not For Use In Diagnostic Procedures.
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