What should be done if the background of T cell typing is high using flow cytometry antibodies from domestic biological testing brands?

The staining system can also amplify background. Control the cell number at around 1×10^6/100 μL, use PBS containing 0.5-1% BSA or 2% FBS as the staining buffer, incubate at 4°C and wash twice, and do not use too low a centrifugal force. Before running the samples, first adjust the voltages using unstained cells and single-stained tubes, and confirm that the negative population is below 10^2; incorrect compensation settings can also make the negative population appear to have high background. If the background is still high after these steps, then suspect that the antibody itself is nonspecific, and change the clone or the fluorescent label. You may ask the supplier to provide flow cytometry data for that clone, but testing it yourself is the most reliable.

If the spleen single-cell suspension undergoes insufficient red blood cell lysis, has many dead cells, and contains many debris, the background of the negative population is easily elevated. After red blood cell lysis, it is recommended to pass through a 70 μm mesh, and the viability by trypan blue counting should preferably be greater than 90%. During staining, add a viability dye, such as 7-AAD or a fixable viability dye, and gate on live cells first during acquisition. Dead cells nonspecifically bind to many fluorescent antibodies; if this layer is not addressed, switching to imported antibodies may still result in high background. You can stain the same sample in parallel with domestic and imported antibodies while adding a viability dye, so that the background difference is comparable.

The mouse spleen cells show high background. It is recommended not to simply reduce the antibody concentration first, but to add Fc Block. The spleen contains many B cells, macrophages, and DCs, with abundant Fc receptors, which can cause increased background with both domestic and imported antibodies. It is advised to use anti-mouse CD16/32 monoclonal antibody, adding 1-2 μg per 1×10^6 cells, pre-block at 4°C for 10-15 minutes, using PBS containing 2% FBS or 1% BSA as the blocking buffer, and then stain for CD3/CD4/CD8. If Fc Block is not performed, directly comparing with imported antibodies is unfair, as much of the background is contributed by Fc receptor or dead cell adsorption, and not necessarily nonspecific binding of the antibody itself.

1: 100 is only a reference working concentration, and the optimal concentration may vary greatly for different brands or labeled batches of the same clone. Suggest using spleen cells for titration: 1:50, 1:100, 1:200, 1:400 points, respectively, to observe the MFI of positive and negative groups, and choose the concentration with the highest signal-to-noise ratio instead of the brightest positive group. The common reason for high background is high concentration or residual free fluorescein. When the background is reduced to 1:200-1:300, sometimes the background is significantly improved and the positive group can still be separated. Simultaneously setting up FMO controls, do not rely solely on same type controls, as the combination of same type and background may not necessarily be consistent.