Accuri c6 cytometer

Expression of PD-L1 was assessed using anti-B7H1-phycoerythrin (PE) (R&D Systems, Minneapolis, MN, USA) at a concentration of 0.05 μg/ml. A PE-conjugated control Ig isotype was used to assess non-specific binding. Briefly, cells were harvested and incubated with the antibodies mentioned above for 30 min in the dark at 4 °C, washed and then analyzed with a BD Accuri™ C6 Cytometer (BD Biosciences, New Jersey, USA). Cleaved caspase-3 immunostaining was performed as previously described^{23 (link)}. Apoptosis was assessed by two methods: propidium iodide (PI) incorporation to analyze the DNA content in permeabilized cells^{24 (link)}, and the annexin V-binding assay in double staining with PI, according to Vermes et al.^{25 (link)}. Cells were analyzed with a BD Accuri™ C6 Cytometer (BD, Becton Dickinson).

For BrdU flow cytometry, an APC BrdU flow kit (BD Biosciences, San Jose, CA, USA, cat #552598) was used following the manufacturer’s protocol. Briefly, cells were incubated with 1mM BrdU in fully supplemented media for 24 h. Cells were then fixed, washed, and analyzed on an Accuri C6 cytometer (BD Biosciences, San Jose, CA, USA) using a flow rate of 35 µL/min, and 50,000 events were collected.
For annexin V flow cytometry, the BD Pharminogen APC Annexin V kit (BD Biosciences, San Jose, CA, USA, cat #550475) was used following the manufacturer’s protocol. Following trypsinization and two washes with cold PBS, 1 × 10⁶ cells were suspended in 1 mL of 1× binding buffer. Cells were then transferred into a new culture tube, and 5 µL of both APC Annexin V and 7-AAD were added to the solution along with 400 µL of 1× binding buffer. Samples were analyzed using an Accuri C6 cytometer (BD Biosciences, San Jose, CA, USA) using a flow rate of 35 µL/min, and 25,000 events were collected. Experimental controls consisted of an unstained sample, a sample treated with 5 µL of APC Annexin V only, and a sample treated with 5 µL of 7-AAD only.

For the murine cell lines, a FITC BrdU flow kit (BD Biosciences, San Jose, CA, cat #559,619) and for the human cells an APC BrdU flow kit (BD Biosciences, San Jose, CA, cat #552,598) were used following the manufacturer’s protocol. The APC kit was required for the human cell lines as MDA-231EV cell lines express GFP. Briefly, cells were incubated with 1 mM BrdU in fully supplemented media for 45 min. Cells were then fixed, washed and analyzed on an Accuri C6 cytometer (BD Biosciences, San Jose, CA) using a flow rate of 35 μl/min and 20,000 events were collected. For the H3K27me3 analysis, cells were fixed in 4% paraformaldehyde for 15min and then permeabilized in 90% methanol for 10 min. Cells were then incubated with a 1:200 dilution of anti-H3K27me3 (Cell Signaling cat #9733S) or IgG KP isotype control (Cell Signaling cat #3900s) for 1 h followed by a 30 min incubation with the appropriate secondary antibody (Alexa Fluor 488 for the mouse cells and APC for the human cells). Cells were then resuspended in 5% 7-AAD solution for 5 min. Cells were analyzed on an Accuri C6 cytometer (BD Biosciences, San Jose, CA) using a flow rate of 35 μl/min and 20,000 events were collected.

After incubation with machilin D for 24 h, the cancer cells were harvested and dissociated using 1 × trypsin/EDTA. We used a previously described method [24 (link)]. A total of 1 × 10⁶ cells were cultured with anti-CD44-FITC and anti-CD24-PE antibodies (BD, San Jose, CA, USA) on ice for 30 min. The cancer cells were centrifuged and washed three times with a 1 × FACS buffer and analyzed by an Accuri C6 cytometer (BD, San Jose, CA, USA). The ALDH1 activity was assayed using an Aldefluor^TM assay kit (StemCell Technologies, Vancouver, BC, Canada). We used a previously described method [24 (link)]. The breast cancer cells were treated with machilin D (50 µM) for 24 h and reacted in ALDH assay buffer at 37 °C for 20 min. The ALDH-positive cells were examined by an Accuri C6 cytometer (BD, San Jose, CA, USA).

Annexin V/propidium iodide (PI) staining was performed using an apoptosis kit from Affimetrix, eBioscience, according to the manufacturer's protocol. Cells were analyzed 24 h to 48 h post-IR by flow cytometry (BD Accuri^TM C6 cytometer) and the flow cytometry data were analyzed by the BD Accuri C6 software.