Caspace fitc vad fmk

To determine ΔΨ_m and caspase activity after irradiation T98G and U251 cells were transfected with TRPM8- or nt siRNA, irradiated (48 h after transfection, 0 or 4 Gy) and incubated for further 24 h in the respective cell culture media. To determine ΔΨ_m, cells were trypsinated, washed and incubated for 30 min at room temperature in Ca²⁺-containing NaCl solution solution (see above) containing the ΔΨ_m-specific dye tetramethylrhodamine ethyl ester perchlorate (TMRE, 25 nM for 30 min, Invitrogen, Karlsruhe, Germany). To assess caspase activity, cells were detached and incubated with CaspACE ™ FITC-VAD-FMK (5 μM for 30 min in cell culture medium, Promega, Mannheim, Germany). TMRE and CaspaACE-specific fluorescence was measured by flow cytometry (FACS Calibur, Becton Dickinson, Heidelberg, Germany, 488 nm excitation wavelength) in fluorescence channel FL-2 (585/42 nm) and FL-1 (530/30 nm) emission wavelength, respectively.

The activity of caspases was determined by using the CaspACE™ FITC-VAD-FMK (Promega, Santiago, Chile). Briefly, cells were treated with the analysed compounds (0 and 25 µM) for 48 h. The cells were incubated with CaspACE™ FITC-VAD-FMK in darkness for 20 min at room temperature. Then, the medium was removed and cells were washed twice with PBS. Exposed cells were collected by tripsinization and centrifugation (10 min at 1500× g). The supernatant was discarded and the cells were re-suspended in PBS and analyzed by flow cytometry using the filter FL3. Results are expressed as percentage of cells stained with CaspACE™ FITC-VAD-FMK [50 (link)].

Spleens were isolated from Sprague Dawley rats at various times post-treatment and post-challenge. Splenocytes were isolated as described above. CD4 and CD8 T-cells were separated with negative selection using CD4 and CD8 T-Cell Isolation kits (StemCell Technologies, Vancouver, BC, Canada). The cross-contamination purity of a typical preparations of CD4+ and CD8+ lymphocytes is shown in Figure S4. N1-S1 cells were labelled with CellTrace Violet (ThermoFisher Scientific, Waltham, MA, USA) according to manufacturer’s instructions. Various ratios of T-cells to labelled N1-S1 cells (5:1, 10:1 and 30:1) were incubated together at 37 °C, 5% CO₂ for 24 h. The cells were harvested and stained with Annexin-V (APC) (ThermoFisher Scientific, Waltham, MA, USA) in annexin binding buffer (1× PBS, 10 mM Hepes, 150 mM NaCl, 2.5 mM CaCl₂) and CaspACE FITC-VAD-FMK (Promega, Madison, WI, USA) at room temperature for 20 min. The cells were washed and propidium iodide was added before running on flow cytometry. Cytotoxicity was indicated.

For determination of apoptosis using FACS analysis^{10 (link),12 (link)}, control and treated H9c2 cells were detached from the 6-well plates by trypsin/EDTA treatment. Supernatants containing floating cells were collected and reunited with detached adherent cells. After centrifugation, cell pellets were re-suspended in culture medium. Cells were counted and apoptosis was determined by Annexin V (BD Pharmingen, Heidelberg, Germany) and CaspACE FITC-VAD-FMK (Promega, Mannheim, Germany) labeling according to the manufacturers’ instructions.
Briefly, 10⁵ cells were incubated with either 5 µL Annexin V-FITC dye in 100 µL Annexin binding buffer for 15 min at room temperature in the dark or with 5 µL of the 1:100 diluted CaspACE FITC-VAD-FMK in 100 µL culture medium for 20 min at 37 °C. The unbound antibodies or dyes were removed by washing the cells with 3 mL Annexin binding buffer or with FACS buffer, respectively.
Data from 5.000 cells were analyzed on a FACS Calibur flow cytometer (BD, Franklin Lakes, NJ, USA). Cell debris was excluded from the measurement by setting gates for intact and apoptotic cells. The data were analyzed by Cell Quest software (BD Biosciences, Franklin Lakes, NJ, USA).

Percentage of cells showing Caspase 3 activation was determined by FITC-VAD-FMK staining, followed by flow cytometry quantification as previously described [46 (link)]. Briefly, both floating death cells and attached cells were collected, centrifuged 300 × g and resuspended in 5 µM FITC-VAD-FMK (CaspACE™ FITC-VAD-FMK, Promega) containing serum-free media and incubated at 37 °C for 30 minutes protected from light. Cells were washed once with PBS, resuspended in PBS containing 0.5 µg/mL of PI (Biolegend, CA USA) and incubated at room temperature for 10 min protected from light. Samples were acquired and analysed with a BD Accuri^TM C6 Flow Cytometer (MA, USA). FITC-VAD-FMK fluorescence was exited using a 488 nm laser and detected with the FL1 525/25 nm filter. PI fluorescence was excited using a 488 nm laser and detected with the FL2 585/40 nm filter.