What fluorescence does CD45RA and CCR7 antibody not fight in the human PBMC multi-color scheme?

Within two weeks of delivery, choose regular fluorescent products that are available in stock domestically. When applying for an order, directly ask the agent to check the "spot, batch number, and shipping warehouse" instead of just asking for the catalog price. 50 test specifications, FITC, PE, PerCP-Cy5.5, APC, APC-Cy7 fluorescence are usually more common, and non special dyes will be faster. If the delivery time of an antibody exceeds two weeks, different clones of the same target or different fluorescent substitutions can be considered, but FMO validation is required. Let the agent lock the inventory first, and then conduct experiments; Prepare one or two alternative item numbers at the same time to avoid the impact of single brand out of stock.

The core of a five-color panel that doesn't clash is to assign bright fluorophores to weakly expressed antigens. CCR7 on PBMC T cells has separable positive/negative peaks but generally moderate MFI, so PE is preferred; CD45RA shows clear separation, and FITC is sufficient. CD3, CD4, and CD8 are all strongly expressed and can be assigned to PerCP-Cy5.5, APC-Cy7, and APC. Do not readily assign CCR7 to PerCP-Cy5.5 or APC-Cy7, as background and compensation will be challenging. Another alternative is CD45RA-APC and CCR7-PE, but this occupies APC, and CD8 would need to be moved to APC-Cy7, resulting in greater compensation.

Once the panel is finalized, single-stain compensation and FMO must be performed. In particular, for APC and APC-Cy7, as well as PE and FITC, the voltages and compensation on the Canto II need to be readjusted. It is recommended to first stain one tube of five-color full staining with healthy human PBMC to check whether the proportions of each population are consistent with the literature: CD45RA+CCR7+ naïve, CD45RA-CCR7+ central memory, CD45RA-CCR7- effector memory, etc. If the CCR7-positive peak cannot be resolved, prioritize adjusting the PE voltage and antibody amount rather than changing the dye. Adding a viability dye to the panel may be more robust, but you currently only have five colors, so if the samples are fresh, it can be temporarily omitted.

When Canto II only has 488 and 633 lasers, FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, or APC-Cy7 are preferred for five colors. In the memory T classification, the CCR7 signal is not particularly strong. It is recommended to give CCR7 to PE and CD45RA to FITC, which are classic combinations with relatively controllable compensation. The remaining CD3, CD4, and CD8 can be treated with PerCP-Cy5.5, APC, and APC-Cy7. I commonly use CD45RA-FITC, CCR7-PE, CD3 PerCP-Cy5.5, CD8-APC, CD4-APC-Cy7. Although APC and APC-Cy7 are the same laser, they can be used after adjusting the voltage and compensation. Confirm the spot item number in advance.