Does different CD4-FITC antibody clone numbers affect mouse spleen cell subpopulations?

Both GK1.5 and RM4-5 can recognize mouse CD4, but differences in epitope and affinity may indeed lead to differences in staining intensity. Conventional mouse spleen cells are 10 ^ 6/tube, and two clones can usually separate CD4+and CD4-; The differences are more reflected in peak position, background, and non-specific binding. Suggest using the same sample of spleen cells for gradient titration and comparing the staining index SI instead of just looking at MFI. I use RM4-5 a lot here, and the surface dyeing background is relatively clean; GK1.5 has also been widely used in literature after fixed membrane rupture. Within a budget of 1500 RMB, we can cover 100 tests of imported or domestic products. We need to conduct a trial equipment comparison before making a decision.

For routine mouse splenocyte CD4 subsetting, either clone can be selected, with priority given to in-stock availability, lead time, and channel. In practice, remember to wash twice after red blood cell lysis, add CD16/32 block, and start at 0.5-1 μg/test for 10^6 cells. I suggest requesting two trial-size vials first and running three groups at 1:50, 1:100, and 1:200 to see whether the CD4-positive population percentage matches expectations. If you are only assessing the CD4+ T cell percentage, stability matters more than the maximum signal. For domestic products, if the COA data are complete and inter-batch CV is low, 100 tests can be purchased for under 1500; use small imported sizes as controls.

If only CD4 single labeling is done, FITC compensation pressure is not significant; But if PE, PerCP, etc. are added later, the clone number will cause a shift in the positive peak, which will affect the compensation matrix. It is recommended to use the same clone antibody as a single staining tube when making multi-color clones, and not mix clones. The RM4-5 positive peak is usually concentrated, making it easier to perform routine lymph node grouping; GK1.5 is also stable, but I have encountered a signal drop when used in combination with certain fixed rupture systems. Your budget for a single 100 test does not exceed 1500, and the common spot price of RM4-5 is basically within the range. You can buy a small specification and run a titration first.

Staining index and compensation differences are jointly affected by clone, F/P ratio, and storage state, and cannot be judged by clone number alone. For FITC in splenocytes, pay attention to autofluorescence; it is recommended to perform Fc Block first, then titrate with a series of 0.25, 0.5, 1, and 2 μg/test. Record the positive peak MFI, negative peak MFI, and SD at each point, and calculate the SI; choose the concentration with a high SI and no elevation of the negative peak. I once encountered weak staining of splenocytes with GK1.5 at 1:100; after reducing to 1:25, separation improved, so you must do it yourself. Keep the volume for 10^6/tube within 100 μL; do not let the antibody be diluted.