Flow cytometric antibodies

Oral squamous carcinoma stem cells (OSCSCs) were isolated from patients with tongue tumors at the University of California, Los Angeles (UCLA) [11 (link)] and were cultured in RPMI 1640 (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gemini Bio-Product, CA, USA). RPMI 1640 supplemented with 10% FBS was used to culture human NK cells and monocytes. Anti-CD16 mAbs, and flow cytometric antibodies were purchased from Biolegend (San Diego, CA, USA). Recombinant human IL-2 was obtained from Hoffman (La Roche, NJ, USA). AJ2 is a combination of seven different strains of gram-positive probiotic bacteria: Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus casei. AJ2 were sonicated as described previously [5 (link)]. RPMI 1640 supplemented with 10% FBS was used to re-suspend sonicated AJ2 (sAJ2). Human ELISA kits for IFN-γ were purchased from Biolegend (San Diego, CA, USA). Phosphate buffered saline (PBS) and bovine serum albumin (BSA) were purchased from Life Technologies (Carlsbad, CA, USA).

The dissociated cells were washed and incubated with flow cytometric antibodies (BioLegend, San Diego, CA). IgG2b was used isotype control. After 30 min incubation, the antibody-stained cells were washed and analyzed by flow cytometry (EPICS XL-MCL, Coulter, Miami, FL). The data were evaluated on a computer software package (FlowJo vX, Flowjo, Ashland, OR).