Goat polyclonal anti rabbit igg h l alexa fluor 488

Harvested cells were washed in PBS buffer and distributed into 96 well plates (5 × 10⁵ cells/well), washed in 200 μL FACS buffer (PBS + 1% BSA), pelleted at 400× g (in a Megafuge 2.0R, Heraeus centrifuge, Labcare, Buckinghamshire, UK) for 4 min and fixed in 200 μL Foxp3/Transcription Factor Fix/ Perm solution for 45 min at room temperature. Cells were washed twice in 200 μL 1x Permeabilization/Wash buffer (PBS, 1% BSA, 0.5% Saponin), pelleted and resuspended in primary antibody solution (50 μL rabbit anti-8OH-dG polyclonal antibody or isotype control (diluted at 400 ng/mL in 1x Perm/Wash buffer) at 4 °C overnight, washed twice in Perm/Wash buffer and resuspended in secondary antibody solution (Goat Polyclonal Anti-Rabbit IgG H&L (Alexa Fluor^® 488), Abcam) at 1000 ng/mL) for an hour at room temperature. Cells were then washed twice in Perm/wash buffer and resuspended in 400 μL FACS buffer for analysis by flow cytometry using a BD FACSCanto II (Becton Dickinson, San Jose, CA, USA) and FlowJo software (TreeStar, Ashland, OR, USA).

The drugs, including Cln and GCV, were purchased from Sigma-Aldrich (St. Louis, MO, USA), and ICI 118551 hydrochloride (ICI) was purchased from Tocris Bioscience (Bristol, UK). The antibodies, including rabbit monoclonal anti-S100-β, rabbit polyclonal anti-Cx43, goat anti-rabbit IgG H&L (HRP), and goat polyclonal anti-rabbit IgG H&L (Alexa Fluor 488) were bought from Abcam (Cambridge, MA, USA). The rabbit polyclonal primary antibody against cleaved caspase-3 was purchased from Cell Signaling Technology (Beverly, MA, USA), anti-Bax, anti-Bcl-2, and anti-TK were bought from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The rabbit polyclonal anti-β-actin was obtained from PADZA (Tehran, Iran). DMEM/F12, fetal bovine serum (FBS), antibiotic/antimycotic, trypsin-EDTA, and MTT were purchased from Gibco Life Technologies (Carlsbad, CA, USA). The Annexin V/7-AAD detection kit, TRIzol reagent, and the reverse transcription kit were procured from BioLegend (San Diego, CA, USA), Thermo Fisher Scientific (Waltham, MA, USA), and Takara (Shiga, Japan), respectively. The enhanced chemiluminescence (ECL) kit was bought from Amersham Biosciences (Freiburg, Germany).