Af488 conjugated anti parp 1

Relative expression of PARP isoforms was determined using flow cytometry in U87MG cells after exposure to unlabelled olaparib. U87MG cells (1 × 10⁶ cells/well) were seeded in 96-well plates and exposed to olaparib (final concentration: 0–1 μM in 200 μL growth medium) for 3 h at 37 °C. Cells were washed with FACS buffer (PBS, 2% FBS, 1 mM EDTA, 0.1% NaN₃) and centrifugation at 500 × g for 5 min. Immunostaining was performed using the Foxp3/transcription factor staining buffer set (eBioscience™, USA). Intracellular staining was conducted in permeabilisation buffer for 30 min at 4 °C in the dark separately using the following antibodies: AF488-conjugated anti-PARP-1 (1:100; sc-80070), AF594-conjugated anti-PARP-2 (1:100; sc-393310) and AF488-conjugated anti-PARP-3 (1:100; sc-390771) from Santa Cruz Biotechnology Inc. Fixable viability dye ef780 (1:4000; eBioscience™; 65-0865-14) was used for live and dead cells discrimination. Fixation of immunostained cells was performed for 15 min at room temperature. Flow cytometry was conducted on the CytoFLEX benchtop flow cytometer (Beckman Coulter, USA), with appropriate laser and filters, positive and negative controls. Data were analysed using FlowJo™ (Tree Star Inc., BD Biosciences, USA).

Relative expression of PARP1, 2 and 3, ALDH2 and IMPDH2 was determined by flow cytometry. Cells (1 × 10⁶ cells/well) were seeded in 96-well plates in growth medium, washed with FACS buffer (PBS, 2% FBS, 1 mM EDTA, 0.1% NaN₃) and centrifuged at 500×g for 5 min. Immunostaining was performed using the Foxp3/transcription factor staining buffer set (eBioscience^TM, USA). Intracellular staining was conducted in permeabilisation buffer for 30 min at 4 °C in the dark using the following antibodies: AF488-conjugated anti-PARP-1 (1:100; sc-80070), AF594-conjugated anti-PARP-2 (1:100; sc-393310), anti-PARP-3 (1:100; sc-390771) or anti-PARP-tankyrase-1/2 (1:100; sc-365897) from Santa Cruz Biotechnology Inc. (USA), AF488-conjugated anti-IMPDH2 (1:500; ab-200770) from Abcam plc. (UK) and AF488-conjugated anti-ALDH2 (1:100, ABIN6817568) from Antibodies-online GmbH (UK). Fixable viability dye ef780 (1:4000; eBioscience^TM; 65-0865-14) was used for live and dead cells discrimination. Fixation of immunostained cells was performed for 15 min at room temperature. Flow cytometry was conducted on the CytoFLEX benchtop flow cytometer (Beckman Coulter, USA), with appropriate lasers and filters, positive and negative controls. Data were analysed using FlowJo^TM (Tree Star Inc., BD Biosciences, USA).