Cyan adp facs machine

For all FACS analysis, treated breast cancer cells were harvested using Accutase (BD Biosciences; San Jose, CA, USA) in place of trypsin–EDTA. Cells (1 × 10⁶) were then suspended in 100 µl staining buffer and placed in microcentrifuge tubes.
Phycoerythrin (PE)-conjugated mouse anti-human CD24 (20 µl) and allophycocyanin (APC)-conjugated mouse anti-human CD44 (20 µl) antibodies (both from BD Biosciences) were added to each sample, along with the necessary FACS dye controls, and the samples incubated on ice for 45 min. Cells were washed twice in staining buffer, resuspended in 500 µl staining buffer and 1 μl of 250 μg/ml PI, and subjected to FACS analysis.
Aldehyde dehydrogenase (ALDH) activity was assessed using the ALDEFLUOR kit (STEMCELL Technologies Inc.; Vancouver, BC, Canada), according to the manufacturer’s protocol. All samples were processed within 15 min of the final wash. Cells were visualized using a Beckman Coulter CyAn ADP FACS machine running Summit 5.2 software and results analyzed as previously described (Ginestier et al. 2007 (link)).

Cells were trypsinized and collected together with medium after being exposed to CS or fresh air. The cells were centrifuged (660× g for 5 min) and the medium was removed. The cells were fixed in Paraformaldehyde (4% in DDW) for 10 min then washed with PBS without Ca⁺² and Mg⁺². The cells were resuspended in 800 μL of PBS containing 0.2% Tween (PBS-T) for 10 mins on ice. The cells were washed again with PBS and resuspended in 100 μL of PBS-T containing 3% BSA and anti-TSPO or anti-cAMP antibodies (1:100) (Abcam, Cambridge, UK). The resuspended samples were incubated overnight at 4 °C. On the next day, the cells were washed and resuspended in 100 μL of PBS-T containing 3% BSA and Alexa Fluor 488 AffiniPure Goat Anti-Rabbit IgG (1:1.000) (Abcam, Cambridge, UK). Mean fluorescence intensity (MFI) was measured using fluorescence assisted cell sorting (FACS) with a CyAN ADP FACS machine (Beckman Coulter, Brea, CA, USA). The data were analyzed using FlowJo software (10th version, FlowJo LLC, Ashland, OR, USA).