|Product Includes||Quantity (with Count)||Reactivity||Dilution|
|CD3 (UCHT1) Mouse mAb (PE Conjugate) 46233||1 x 500 µl||H|
|CD4 (RPA-T4) Mouse mAb (violetFluor™ 450 Conjugate) 26755||1 x 500 µl||H|
|CD8α (RPA-T8) Mouse mAb (PE-Cy7® Conjugate) 27442||1 x 500 µl||H|
|TNF-α (D1G2) Rabbit mAb (Alexa Fluor® 647 Conjugate) 60959||1 x 100 µl||H|
|IFN-γ (D3H2) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) 12942||1 x 100 µl||H|
All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Triton™ X-100) #51995, or individually using the catalog numbers listed below.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
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.
NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.
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.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or Triton™ X-100. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. Conduct a small-scale experiment if you are unsure.
NOTE: Count cells using a hemocytometer or alternative method.
posted January 2017
revised June 2020
Protocol Id: 1344
CD3 (UCHT1) Mouse mAb (PE Conjugate), CD4 (RPA-T4) Mouse mAb (violetFluor™ 450 Conjugate), and CD8α (RPA-T8) Mouse mAb (PE-Cy7® Conjugate) are supplied in 10 mM NaH2PO4, 150 mM NaCl, 0.09% NaN3, 0.1% gelatin, pH7.2. TNF-α (D1G2) Rabbit mAb (Alexa Fluor® 647 Conjugate) and IFN-γ (D3H2) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) are supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. 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.
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.
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