Alexa fluor 594 goat anti rat igg

A total of 60,000 G₀-arrested MCF10A cells grown on cover slips were irradiated with 10 Gray IR and then allowed to recover for 3 hr at 37 °C with 5% CO₂. Cells were then washed with PBS containing 0.1% Tween-20 (PBST), pre-extracted using cold 0.5% Triton-X100 in PBS for 5 min, fixed with 4% formaldehyde for 15 min, and blocked in 3% BSA-PBST for 1 hr at room temperature. Cells were incubated overnight at 4 °C in primary antibody (anti-RPA32, Cell Signaling Technology, #2208). Samples diluted in 3% BSA-PBST were then washed 3 x with PBST, incubated with secondary antibody diluted in 3% BSA (Alexa Fluor 594 Goat anti-Rat IgG, BioLegend, #405422) in the dark for 1 hr at room temperature, washed 3 x with PBST, and mounted in Prolong Gold Antifade Mountant with DAPI (Life Technologies, #P-36931). Images were taken using a Biotek Lionheart Automatic Microscope and foci quantification was performed using Biotek Gen5 software.

InVivo MAb anti-mouse 4-1BB (clone: 3H3) and InVivoPlus rat IgG2a isotype control (clone: 2A3) were supplied by BioXCell (New Hampshire, USA). IRDye® 680RD NHS Ester Infrared Dye was supplied by Li-cor (Nebraska, USA). Fixable Viability Stain 780 was purchased from BD Pharmingen (San Diego, USA). Dimethyl sulfoxide (DMSO) Hybri-Max (TM) sterile-filtered BioReagent was purchased from Sigma (St. Louis, MO, USA). The AbC™ total antibody compensation bead kit was acquired from Thermo Fisher (Waltham, MA, USA). PE anti-mouse CD4, FITC anti-mouse CD8a, APC anti-mouse CD137, and Alexa Fluor 594 goat anti-rat IgG (minimal x-reactivity) antibodies were supplied by BioLegend (San Diego, CA, USA). Anti-rabbit IgG (H + L), F(ab')2 fragment (Alexa Fluor® 488 Conjugate) was purchased from Cell Signaling Technology (Massachusetts, USA). A purified anti-mouse CD3 antibody was supplied by Thermo (Waltham, MA, USA). An anti-CD137 antibody was purchased from Abcam (Cambridgeshire, UK).