Guava easycyte plus flow cytometer

Cells were treated with BI 6727 (BT16 IC50=50nm; BT12 and MAF-A737 IC50= 24 nm), and then allowed to grow in normal culture medium for 24, 48 and 72 hours. The cell concentration was determined following staining with Guava ViaCount reagent (Millipore, Billerica, MA). Equal numbers of cells were then stained using Guava Nexin reagent (Millipore) to detect apoptotic cells. Samples were run on a Guava EasyCyte Plus flow cytometer (Millipore).

As previously described, inducible GFP plasmids were transformed into S. Typhimurium strains in order to assess both Salmonella-induced cell death and invasion by flow cytometry (24 (link)). Briefly, bacterial cultures were prepared as described above and used to infect LCLs and THP-1 monocytes (multiplicity of infection [MOI], 10 or 30), as well as HeLa cells (MOI, 5). For experiments with nonmotile ΔflhDC mutants, cells were centrifuged at 500 × g for 10 min to enable infection. At 1 h postinfection, cells were treated with gentamicin (50 μg/ml), and IPTG (isopropyl-β-d-thiogalactopyranoside) was added at 2 h postinfection to induce GFP expression. At 3.5 h postinfection, cells were assessed for cell death using 7-aminoactinomycin D (7-AAD; Biomol), with death being read by a Guava EasyCyte Plus flow cytometer (Millipore). Percent invasion was determined by quantifying the number of GFP⁺ 7-AAD⁻ cells at 3.5 h postinfection using the Guava EasyCyte Plus flow cytometer. Gates were set by using uninfected cells to gate out GFP-negative (GFP⁻) cells and by using the natural break in 7-AAD⁻ and 7-AAD-positive (7-AAD⁺) cells (Fig. 1A).

Annexin V/7-AAD staining method was used to identify and quantify apoptotic cells. SCC-25 cells (2 × 10⁵–5 × 10⁵ cells/mL) were treated according to the manufacturer’s instruction of ANNEX V Kit. The ability of different treatments to induce apoptosis against SCC-25 cells was assessed by flow cytometry using the Muse^® Annexin V and Dead Cell Assay kit (EMD Millipore Corporation). The extent of apoptosis was quantified according to the percentage of Annexin V-positive cells. The ability of the different treatments to induce apoptosis against tongue squamous carcinoma cells was assessed for this analysis. SCC-25 cells were plated in 24-well plates at a density of 5 × 10⁴ cells per well. After 24 h of incubation, cells were incubated with various concentrations of CBD, andrographolide, cisplatin, and 5-FU (at 0, 10, 30, 100, and 300 µM) for 24 h. After 24 h of treatment, cells were detached by trypsin and then centrifuged at 1200 rpm for 10 min. After centrifugation, 1 × 10⁵ cells were stained according to the manufacturer’s instructions and analyzed using Guava easyCyte Plus Flow Cytometer (Millipore, Billerica, MA, USA).

SUM159 and DU145 cells were labeled using the PKH26 Red Fluorescent Cell Linker Kit (Sigma) according to the manufacturer’s instructions. The labeled SUM159 cells were treated with doxorubicin (1 µg/ml) to generate chemotherapy enriched dormant cells, as described above. Likewise, PKH26-labelled DU145 were treated with docetaxel (10 nM) to generate chemotherapy-enriched dormant cells, as described above. Labelled cells were detected using the Guava EasyCyte Plus flow cytometer (Millipore).