Mouse Progenitor Exhausted CD8+ T Cell Markers Flow Cytometry Panel #60395
Important Ordering Details
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Protocol
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.
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.
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.
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.
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.
限制使用
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For Research Use Only. Not For Use In Diagnostic Procedures.
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