Which flow cytometer antibody can stably isolate Foxp3 after membrane rupture?

Small package trials can be applied for. You have 40 samples, and 25 tests may not be enough. It is recommended to ask for the specifications of 50 tests. Imported and domestic manufacturers usually provide trial equipment or small specifications for laboratory evaluation. When applying, clearly state the instrument model (several lasers), sample quantity, current brand of fixed membrane rupture kit, previous clone number, and issue, and ask the other party to recommend a compatible Foxp3 clone. Do not change both the antibody and membrane lysis kit at the same time, otherwise you will not know where the variables are. First, use the existing system to parallelly stain two or three candidate antibodies, selecting those with low background and good clustering.

The most commonly used clone FJK-16s for mouse Foxp3 flow cytometry can clearly group in most cases in a fixed membrane breaking system; MF-14 and MH-4 are also used. You were not clearly grouped before, please confirm if the clone number matches the fixed membrane lysis kit first. Different membrane breaking systems have different effects on antigen epitopes. It is recommended to fix the membrane and perform gradient titration using the same clone, ranging from 0.25 to 2 μ g/test, to compare the distance between positive/negative peaks. If the overall positive peak is weak but the background is low, it may be due to insufficient concentration or insufficient membrane rupture; If the background is high, consider insufficient blocking or non-specific antibodies.

High background in Foxp3 nuclear staining makes fixation and permeabilization time critical. Perform viability testing on the spleen single-cell suspension first, as a high number of dead cells will adsorb antibodies. It is recommended to block with CD16/32 before fixation, thoroughly wash away unbound antibodies after surface staining for CD4/CD25, and then fix and permeabilize. Commercial Foxp3 buffer generally requires fixation for 30-60 minutes, and after permeabilization, staining for 30-60 minutes at 4°C in the dark. Prepare the staining solution with permeabilization buffer, not ordinary FACS buffer. For 40 samples, keep the processing time as consistent as possible, and set the same controls when staining in batches.

If switching antibodies still results in poor separation, check the gating and controls. It is recommended to use FMO (CD4/CD25 surface staining, Foxp3 empty) and isotype controls to help with gating. For spleen Treg percentage, generally look at Foxp3+ within CD4+, with CD25 as an auxiliary marker. During analysis, first exclude debris and doublets, then gate CD4+, and then examine Foxp3. Do not skip titration; after fixation and permeabilization, the optimal antibody concentration may be higher than for surface staining. For trial samples, first test 3 concentrations, then use the optimal concentration to run a sufficient number of samples. Place an order only after stability validation.