Accuri plus flow cytometer

Cells (1x10⁶ cells) were collected by centrifugation, washed with ice-cold methanol for fixation, and then permeabilized with 0.1% Triton-X100 containing 2% bovine serum albumin-phosphate buffered saline (PBS). Following washing with PBS, cells were stained with rabbit anti-Oct3/4, rabbit anti-Nanog, or mouse anti-p53 antibodies. Anti-rabbit-AF543 and anti-mouse-AF488 were used as secondary antibodies. Cells were analyzed with a BD Accuri Plus Flow Cytometer (BD). For 10,000 events, percentages of positive populations were determined by using BD Accuri Plus software (BD).

Differential cell-surface marker expression of CD74 on T84 IECs challenged with the LrS, STS, LrS, and STS or medium controls was determined using a BD Accuri C6 flow cytometer. Following challenge, 1 × 10⁶ cells were resuspended in D-phosphate buffered saline (D-PBS) and cells were stained with the viability dye 7-AAD (Tonbo, 13–6,993) for 10 min on ice while protected from light. Cells were washed with 1 mL of cell staining buffer (CSB) (4% calf serum, 5 mM EDTA in D-PBS) and centrifuged for 5 min at 400 × g (4°C). Immediately following viability staining, non-specific Fc-mediated interactions were blocked by the addition of 100 μL of blocking buffer [10% calf serum (heat inactivated) in D-PBS] to the cell suspension for 10 min. Anti-human CD74 (BioLegend, clone LN2) was added to the cell suspension followed by incubation on ice and protected from light for 30 min. Cells were washed with CSB as described above, resuspended in 100 μL of fixation buffer (4% paraformaldehyde-PBS) and incubated for 30 min at room temperature protected from light. Data acquisition was done using the BD Accuri Plus flow cytometer and corresponding software package. 30,000 viable cells (as determined by incorporation of 7-AAD viability dye [Tonbo 13–6,993)] were acquired for each experiment and subsequent analysis was done using FlowJo v. 10.