[Effects of two incubation protocols on median antibody fluorescence intensity, percentage of positive cells, and group differences in flow cytometry].

Objective This study aimed to compare the effects of two distinct intracellular antibody staining protocols-the sequential antibody incubation (A-P-A) method and the one-step antibody incubation (P-A) method-on flow cytometry results. Specifically, it focused on the median fluorescence intensity (MFI), the percentage of positive cells, and the observed differences between experimental groups. Methods The peripheral blood and splenocytes from 12-week-old male Kunming mice under different treatments were analyzed using two intracellular staining methods to assess differences in immune cell subset proportions, MFI and intergroup variability. Results In murine peripheral blood, the A-P-A protocol demonstrated higher sensitivity for MFI but poorer reproducibility compared to the P-A method. While intergroup trends for MFI were consistent for five antibodies and PerCP-Cy5.5-7-AAD dye, they diverged for four other antibodies. Although cell viability was comparable, three immune cell subsets exhibited opposite intergroup trends between the protocols. Statistical analysis indicated that significant differences in Ly-6c-high monocyte proportions were detected only in the A-P-A method but not in the P-A method, whereas significant differences in M1 macrophage proportions were observed only in the P-A method but not in the A-P-A method. Importantly, the trends across treatment groups were consistent between the two incubation methods for both cell types. In splenocytes, both the nine antibodies and all cell populations demonstrated consistent intergroup trends across the two protocols. The A-P-A method generally produced greater intergroup variability in MFI and specifically enabled the detection of significant differences in cell viability and Ly-6c-low monocytes, while the P-A method revealed a significant difference in M2 macrophage proportions. Conclusion For murine peripheral blood, the A-P-A method shows higher sensitivity but lower reproducibility in MFI and lymphocyte subsets compared to the P-A method. Since statistical analysis reveals no significant advantage for either method, the P-A protocol is recommended due to its procedural simplicity. In splenocytes, both protocols show consistent intergroup trends. The A-P-A method provides greater discrimination in MFI, while both methods are comparable in terms of subset proportions.