Cellevent senescence green flow cytometry assay kit

Using the same experimental design (DHX38-Crimson), we performed beta-galactosidase staining using the CellEvent Senescence Green Flow Cytometry Assay Kit from Invitrogen on day 2, day 4 and day 7 following the manufacturer’s protocol. Briefly, we trypsinized and we fixed the cells with 2% paraformaldehyde solution for 10 minutes at room temperature, washed them in 1%BSA/PBS and incubated for 1h30 in 1/500 working solution. After incubation, we washed the cells with 1%BSA/PBS and analyzed them by flow cytometry. We measured the β-galactosidase fluorescence signal in positive and negative Crimson cells independently. As positive control, non-infected cells were treated with 20 μM of Etoposide (Sigma, E1383-25) for 2, 4 and 7 days.

The β-gal tests were prepared based on the commercial kit (CellEvent Senescence Green Flow Cytometry Assay Kit, Invitrogen) adapted for the bacteria-based experiments. Briefly, after 6 h of stimulation, the cell medium was collected from the wells and the CaCo2/HT29 layer was gently rinsed with warm PBS. Then both fractions were combined and centrifuged (8 min, 3,500 × g). Finally, the bacterial pellet was washed in PBS (8 min, 3,500 × g) and bacteria were suspended in 2% PFA and incubated for 10 min protected from light. Next, the samples were washed twice in PBS (8 min, 3,500 × g) and stained with CellEvent Senescence Green Probe reagent diluted 1:500 in working buffer (1 h, 37 °C, protected from light and without CO₂ additional sources). Then, the samples were washed, suspended in 1 ml of PBS and kept in the fridge before the flow cytometry analysis. The flow cytometry data collection (10,000 events) was based on a blue laser with 488/8 and 610/20 nm filters. The bacterial population was gated as previously.

Isolated human lung cells were stained for SA-β-Gal using the Cell Event™ Senescence Green Flow Cytometry Assay Kit (Invitrogen™, Thermo Fisher Scientific, C10840), following manufacturer’s instructions. Briefly, after dissociation, lung cells were fixed in 2% PFA (Thermo Scientific, 28908) for 10 min at room temperature. Cells were washed, centrifuged, and resuspended in freshly prepared working solution and incubated for 1–2 h at 37 °C in a dry incubator, protected from the light. After incubation, cell suspensions were washed twice with 1% BSA in D-PBS and permeabilized with 0.1% Tween20^® (Sigma, St. Quentin Fallavier, France, P1379) in D-PBS for 15 min on ice. After washing, cells were incubated with anti-LGR6 antibody (1:100; Abcam, Waltham, MA, USA, ab126747) and with APC-conjugated anti-human CD45 and CD31 antibodies (1:1000 and 1:1500; BioLegend, San Diego, CA, USA, 304012 and 303116) for 60 min on ice. Secondary DyLight™ 405 donkey anti-rabbit (1:500; Jackson ImmunoResearch Europe Ltd., 711-475-152) was used to detect LGR6 expression. Samples were run on BD Scientific Canto II Flow Cytometer (BD Bioscience, San Diego, CA, USA, RRID: SCR_018056) and flow cytometry data were analysed with FCS Express™ (version 7, DeNovo Software, Glendale, CA, USA, RRID: SCR_016431).

Cells were harvested at different time points after LACTB induction with doxycycline. For analysis of apoptosis, 1 × 10⁶ cells were stained with Annexin V-Alexa Fluor 488 and propidium iodide (PI) for 15 min in dark, using the Apoptotic cell assay kit (ThermoFisher) following the instructions provided. The signal of 20,000 events was analyzed. Determination of apoptosis was measured using an LSR Fortessa cell analyzer (BD Biosciences) at the IOCB flow cytometry facility, and data was analyzed using FlowJo 10.7.1 software. Data was plotted using GraphPad 8.0 software.
Senescence was analyzed using the CellEvent™ Senescence Green Flow Cytometry Assay Kit (ThermoFisher). β-galactosidase activity was measured by flow cytometry and analyzed as described above.

Cell Event Senescence Green flow cytometry assay kit (Thermofisher Scientific, Waltham MA, USA) was used according to the manufacturer’s instruction. Briefly DCs were first stained for cell surface marker CD11c using regular staining protocol discussed in detail in the following sections. After the last wash, cells were fixed with 4% paraformaldehyde (PFA) for 10 minutes at room temperature followed by washing to remove the fixative solution. Cells were then incubated with Cell Event Senescence Green probe (Thermofisher Scientific, Waltham MA, USA) at 1:500 dilution for 2 hours at 37°C with no CO2. Cells were then washed with PBS, 2% FBS and data acquired by MACSQuant analyzer machine and MACSQuantify software (Miltenyi Biotech Auburn, CA, USA).