Revision 6
Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

FC-FP

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:

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UniProt ID:

#Q02242, #P36402, #P10144, #P22646, #P11942, #P01731

Entrez-Gene Id:

18566, 6932, 3002, 12501, 12502, 12525

Product Information

Product Usage Information

All antibodies in this kit are compatible with the Intracellular Flow Cytometry Kit (TritonTM X-100) #51995 and can be used in a single staining mix on fixed and permeabilized cells. Prior to fixation and antibody incubation, we recommend adding a fixable viability dye such as the Ghost Dye Violet 510 Viability Dye to enable identification and exclusion of dead cells from analysis.

This panel can be combined with peptide-MHC tetramer staining to identify antigen-specific cells. Tetramer staining is often performed on live cells. If tetramer staining is desired, we recommend comparing activity of the reagent in live cell staining vs. the recommended protocol for the panel. If there is not a significant difference between the two conditions, the tetramer can be included in the antibody panel staining mix. If there is significant loss of activity following fixation and permeabilization, tetramer staining should be done on live cells prior to fixation and permeabilization.

Gating strategy for observing progenitor exhausted CD8+ T cells:
If a fixable viability dye was used, first gate on viable cells. Next, gate on lymphocytes based on forward scatter and side scatter. Cytotoxic T cells are the CD3+CD8+ cells within the lymphocyte gate. Next, observe TCF1 and PD-1 expression on the cytotoxic T cells. Cytotoxic T cells that are TCF1+PD-1+ are exhausted progenitor cells and are expected to be mostly negative for Granzyme B expression. Cytotoxic T cells that are TCF1-PD-1+ are differentiated cells with effector potential and are expected to be mostly positive for expression of Granzyme B.

Storage

PD-1 (Intracellular Domain) (D7D5W) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate), TCF1/TCF7 (C63D9) Rabbit mAb (PE Conjugate), and Granzyme B (D2H2F) Rabbit mAb (Alexa Fluor® 647 Conjugate) are supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. CD8α (2.43) Rat mAb (PE-Cy7® Conjugate) and CD3 (17A2) Rat mAb (violetFluor 450 Conjugate) are supplied in 10 mM NaH2PO4, 150 mM NaCl, 0.09% NaN3, 0.1% gelatin, pH 7.2.

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 Mouse Progenitor Exhausted CD8+ T Cell Markers Flow Cytometry Panel detects endogenous levels of its target protein. CD8α (2.43) Rat mAb (PE-Cy7® Conjugate) and CD3 (17A2) Rat mAb (violetFluor 450 Conjugate) detect epitopes within the extracellular domains. PD-1 (Intracellular Domain) (D7D5W) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate), TCF1/TCF7 (C63D9) Rabbit mAb (PE Conjugate), and Granzyme B (D2H2F) Rabbit mAb (Alexa Fluor® 647 Conjugate) detect epitopes within the intracellular domains.

Species Reactivity:

Mouse

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.

Product Description

The Mouse Progenitor Exhausted CD8+ T Cell Markers Flow Cytometry Panel can be used to identify progenitor exhausted CD8+ T cells and their differentiated T cells with effector potential.

Cytotoxic T cells are identified by co-expression of CD3 and CD8α. Among these cells, the exhausted progenitor cells are TCF1+PD-1+ cells and are mostly Granzyme B-; the differentiated cells with effector potential are TCF1-PD-1+ and are mostly GranzymeB+. In response to immunotherapy, these progenitor TCF1+PD-1+CD8+ cells can expand and differentiate into TCF1-PD-1+CD8+ effector cells.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Applications Key

FC-FP: Flow Cytometry (Fixed/Permeabilized)

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

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.

Revision 6
#60395

Mouse Progenitor Exhausted CD8+ T Cell Markers Flow Cytometry Panel

Flow Cytometry Image 1: Mouse Progenitor Exhausted CD8+ T Cell Markers Flow Cytometry Panel Expand Image
Flow cytometric analysis of fixed and permeabilized NIH-3T3 cells (blue, negative) and EL4 cells (green, positive) using PD-1 (D7D5W) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) (solid lines) or a concentration- matched Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 488 Conjugate) #2975 (dashed lines).
Flow Cytometry Image 2: Mouse Progenitor Exhausted CD8+ T Cell Markers Flow Cytometry Panel Expand Image
Flow cytometric analysis of Jurkat cells using TCF1/TCF7 (C63D9) Rabbit mAb (PE Conjugate) (solid line) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (dashed line).
Flow Cytometry Image 3: Mouse Progenitor Exhausted CD8+ T Cell Markers Flow Cytometry Panel Expand Image
Flow cytometric analysis of live mouse splenocytes using CD3 (17A2) Rat mAb (violetFluor 450 Conjugate) (solid line) compared to concentration-matched Rat (LTF-2) mAb IgG2b Isotype Control (violetFluor 450 Conjugate) #52572 (dashed line).
Flow Cytometry Image 4: Mouse Progenitor Exhausted CD8+ T Cell Markers Flow Cytometry Panel Expand Image
Flow cytometric analysis of live mouse splenocytes using CD8α (2.43) Rat mAb (PE-Cy7® Conjugate) (solid line) compared to concentration-matched Rat (LTF-2) mAb IgG2b Isotype Control (PE-Cy7® Conjugate) #43426 (dashed line).
Flow Cytometry Image 5: Mouse Progenitor Exhausted CD8+ T Cell Markers Flow Cytometry Panel Expand Image
Flow cytometric analysis of human peripheral blood mononuclear cells, untreated (left column) or treated with anti-CD3 plus anti-CD28 (10 μg/ml, 72 hr; right column), using Granzyme B (D2H2F) Rabbit mAb (Alexa Fluor® 647 Conjugate) (top row) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 647 Conjugate) #2985 (bottom row), and co-stained with a CD8 Antibody.