What is the starting concentration for flow cytometry antibody titration? Is there a trial installation that can be applied for?

Trial package application: Imported brands such as BioLegend, BD, Thermo, and Miltenyi often have small packages or trial packages, but they need to find authorized agents for an indefinite period; Domestic products such as Sizheng Cypress Elabscience、 Dacheng may also provide 5-10 tests for trial. Suggest applying for two or three companies at the same time, specifying that the equipment, samples, and budget per unit should not exceed 2000. The trial volume is small, and it is not enough to perform a complete titration. You can first do three points at 1:50, 1:100, and 1:200 to determine the approximate working concentration. When comparing, use PBMCs from the same donor on the same day to avoid inter batch and machine state interference.

The recommended starting concentration for titration is the midpoint according to the instructions, and double the series up and down, such as 1:25, 1:50, 1:100, 1:200, and 1:400. CD8a is strong in the PBMC positive group, and APC is also bright. A weak 1:100 ratio may be due to issues with the antibody itself, cell count, or voltage. Titrate with a uniform cell volume of 10 ^ 6/100 μ L, stain on ice for 30 minutes, and make single tubes for each concentration. Calculate the staining index SI=(MFIpos MFIneg)/(2 × SDneg), and select the concentration with the highest SI and no increase in background. If 1:25 is still weak, check the antibody preservation, dilution solution, and whether it is fixed before re staining.

For the formal titration experiment, it is recommended to record the negative peak position of the FMO control to help determine the positive threshold. If 1:100 is weak, and 1:50 shows improved signal with acceptable background, choose 1:50; do not blindly pursue high concentrations. After selecting the concentration, perform a small-scale repeat to check whether the CD8+ proportion is consistent with the literature. APC is prone to photobleaching; after staining, store in the dark and run on the instrument as soon as possible. If two or three trials are unsatisfactory, it may be related to the clone, instrument voltage, or compensation not being properly adjusted. You can ask the manufacturer's technical support to review the data remotely before deciding whether to replace the product.

CD8a-APC staining of the positive population is weak at 1:100. First rule out experimental factors: pay attention to cell number, staining volume, Fc Block, incubation temperature and time. Whole blood and PBMC require different amounts; if using PBMC, the proportion of CD8+ among T cells is about 20-30%, and the positive peak should be obvious. APC is sensitive to fixation; if fixation is performed before staining, the signal will decrease. It is recommended to use fresh samples for surface staining first, then red blood cell lysis/fixation. If all are normal, it may be due to low antibody titer or a batch issue; contact the manufacturer's technical support.