Intracellular staining permeabilization buffer

Flow cytometry was used to detect the effect of lipids on the characteristics of M1 or M2 macrophages and the proliferation of 4T1 cells. Briefly, after incubation of M1 and M2 macrophages with different concentrations of lipids for 48 h, cells were washed twice, detached from 24-well plates by trypsinization and resuspended in a FACS buffer containing Abs for extracellular labeling. To assess the effect of lipids on the expression of M1 or M2 macrophage markers, cells were labeled with anti-CD86 (1 μg/mL), anti-CD68 (1 μg/mL) and anti-CD163 (1 μg/mL) Abs. To assess the effect of lipids and CM+ on 4T1 cell proliferation and migration, cells were labeled with anti-E-cadherin (1 μg/mL) and anti-vimentin (1 μg/mL) Abs.
For intracellular Ab labeling, cells were first fixed in 4% paraformaldehyde (PFA) for 10 min at RT, followed by permeabilization in intracellular staining permeabilization buffer (Biolegend) for 10 min, then incubated with 100 uL intracellular staining buffer containing anti-mouse CD68-FITC Ab (1 μg/mL) or Ki-67 (1 μg/mL) on ice for 45 min. After washing with PBS, samples were resuspended in 100 μL PBA and measured by flow cytometry (BD LSR III, Bioscience). For each sample, at least 10,000 events were acquired and analyzed using FlowJo 10.1 software.