CAR-T Cell (Whitlow/218 Linker) Transduction Efficiency Flow Cytometry Panel

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 a membrane impermeable viability dye such as Propidium Iodide or 7-AAD to enable identification and exclusion of dead cells from the analysis.

For individual product dilutions, refer to panel product datasheet or individual product pages.

Gating strategy for identifying viable, CAR positive T cell subsets: Gate on live cells using Propidium Iodide or 7-AAD as a histogram or scatterplot with forward scatter (FSC). Use a gate set on side scatter (SSC) vs. FSC to exclude cell debris. Singlets may be identified by setting a gate on FSC-H vs. FSC-A. Next, use a plot of CD45 vs. CD3 to identify CD45+ leukocytes, and CD3+ and CD3- subpopulations. Observe Whitlow/218 Linker vs. CD3 expression and set a gate on CD3+Whitlow/218 Linker+ cells, which are CAR+ T cells. Observe CD4 vs. CD8 expression within the gated population to identify CD4+ and CD8+ T cells that are CAR+. Similarly, observe Whitlow/218 Linker vs. CD14 expression within the CD3- gate to identify contaminating monocytes.

CD3 (UCHT1) Mouse mAb (PE-Cy7^® Conjugate), CD4 (RPA-T4) Mouse mAb (FITC Conjugate), CD8α (RPA-T8) Mouse mAb (PE Conjugate), CD45 (HI30) Mouse mAb (redFluor 710 Conjugate), and CD14 (61D3) Mouse mAb (violetFluor 450 Conjugate) are supplied in 10 mM NaH₂PO₄, 150 mM NaCl, 0.09% NaN₃, 0.1% gelatin, pH 7.2. Whitlow/218 Linker (E3U7Q) Rabbit mAb (Alexa Fluor^® 647 Conjugate) is 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.

Each of the lineage marker antibodies in the CAR-T Cell (Whitlow/218 Linker) Transduction Efficiency Flow Cytometry Panel detects endogenous levels of its target protein. CD3 (UCHT1) Mouse mAb (PE-Cy7^® Conjugate) detects an epitope within the extracellular domain of CD3ε protein. CD4 (RPA-T4) Mouse mAb (FITC Conjugate), CD8α (RPA-T8) Mouse mAb (PE Conjugate), CD45 (HI30) Mouse mAb (redFluor 710 Conjugate), and CD14 (61D3) Mouse mAb (violetFluor 450 Conjugate) detect epitopes within the extracellular domains. Whitlow/218 Linker (E3U7Q) Rabbit mAb (Alexa Fluor^® 647 Conjugate) recognizes exogenously expressed levels of scFv-based CARs containing a Whitlow/218 linker.

The CAR-T Cell (Whitlow/218 Linker) Transduction Efficiency Flow Cytometry Panel can be used to identify conventional human T cell subsets that have been engineered to express scFv-based CARs containing a Whitlow/218 linker.

CD45 is a pan leukocyte marker. 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. Monocytes are identified by the expression of CD14. CAR positive cells are identified by the expression of a Whitlow/218 linker.