Fitc donkey anti rabbit igg

Immunofluorescence cell staining and confocal microscopy were carried out as previously described (37 ). Cells were incubated with primary antibodies against γH2AX (ser139; 1:1000 mouse monoclonal IgG, clone JBW301, Millipore) and either RAD51 (goat polyclonal IgG; Santa Cruz Biotech.) or rabbit polyclonal anti-BRCA1 IgG (1:1000 rabbit polyclonal IgG, Santa Cruz Biotech.) at 4°C overnight. After three 5-min washes with PBS, cells were incubated 1 h at 22°C in the dark, with appropriate secondary antibodies at 1:1000 dilutions (bovine anti-goat IgG Alexa Fluor 488 for RAD51, or donkey anti-rabbit IgG FITC and goat anti-mouse IgG Alexa Fluor 594 for γH2AX [Jackson ImmunoResearch]). Cells were washed 3 times in PBS and mounted under coverslips with Prolong Antifade reagent containing DAPI. Images were acquired with an LSM 510 Zeiss confocal laser-scanning microscope with a 63× oil objective. For quantitative analysis, ≥100 cells from each group were chosen at random and nuclei were (a.) counted manually to determine the percent positive for BRCA1 or RAD51 or γH2AX, based on a threshold of ≥5 discrete foci per nucleus; and (b.) quantified from fluorescence images to obtain the total integrated pixel intensity, from which the background was subtracted (perimeter signal per pixel multiplied by total area pixels). Results were averaged from at least 3 biological replicates.

The quantity of CR1 expressed per cell was initially assessed by flow cytometry as described previously (Barilla-LaBarca et al., 2002 (link)). Briefly, cells were harvested by trypsinization (0.05% trypsin; 0.53 mM EDTA for 1 min) and washed in 1% FCS-PBS (FACS buffer). Rabbit polyclonal anti-CR1 Ab (100 μl; 1:25 dilution of 3.8 mg/ml protein A affinity purified IgG) was added and incubated with the cells for 30 min at 4 °C. Following centrifugation (1500 rpm for 5 min; TOMY high speed refrigerated microcentrifuge MX180) and two washes, FITC-donkey anti-rabbit IgG was added (100 μl of a 1:100 dilution; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA; catalog # 711-095-152). After 30 min incubation at 4 °C, cells were resuspended in FACS buffer and analyzed by flow cytometry (10,000 events). Rabbit IgG (protein A affinity purified) and secondary Ab alone served as controls. Time and dose-dependent analysis of the effect of varying trypsin concentrations on CR1 expression showed that addition of 1 ml of 0.05% trypsin for 1 min did not affect the CR1 copy number/cell. The clones employed were designated as follows: CR1–2 m (2 × 10⁶ CR1/cell), CR1–200k (2 × 10⁵ CR1/cell) and CR1–10k (1 × 10⁴ CR1/cell).