Mouse igg1 fitc

Fifty µL of anticoagulated blood from abdominal aorta of rats was incubated with 10 µL of mouse anti-rat monoclonal CD11b-FITC antibody or mouse IgG₁-FITC (Santa Cruz Biotechnology, Santa Cruz, USA) for 15 min in the dark at room temperature. 450 µL hemolysin was added to the mixture, followed by 10 min incubation. After centrifugation at 1200 rpm for 5 min, supernatant was discarded and the pellet was washed with phosphate buffered solution (PBS) buffer once and suspended in 450 µL of PBS buffer for flow Cytometry analysis. Data were analyzed using Cell Quest software (BD Biosciences). CD11b expression levels were presented as mean fluorescent intensity (MFI) of positive cells.

NHDF cells (up to 1 × 10⁶ ml⁻¹) were incubated with 80 µl of 1X PBS containing 10% heat‐inactivated fetal bovine serum (FBS), 0.1% NaN₃ (binding buffer), and 20 µl of FcR Blocking Reagent (Miltenyi Biotec) for 15 min on ice, following manufacturer's instructions (cat. No. 130‐059‐901), then suspended in the binding buffer and incubated for 15 min on ice. Thereafter, the staining was carried out in binding buffer by incubating the cells for 30 min on ice with an in house produced FITC‐conjugated anti‐FITM‐2 monoclonal antibody, or with an unrelated mouse IgG1‐FITC (Santa Cruz Biotechnology; sc‐2866) as a negative control. For competition assays, cells were pretreated with rBAG3 1× (10 μg/ml) and 10× (100 μg/ml), or with bovine serum albumin (BSA) used as an unrelated control, for 30 min on ice and then 20 μg/ml of the FITC‐conjugated anti‐IFITM‐2 monoclonal antibody was added to the mixture and incubated for additional 30 min on ice. After incubation, cells were washed with PBS/2% FBS/0.1% NaN₃, centrifuged for 10 min at 300 g, resuspended in 300 µl of binding buffer, and analyzed by flow cytometry, using FACSVerse Flow Cytometer (BD Biosciences). The antibody FITC‐conjugation was performed using a FluoroTag FITC conjugation kit (FITC1‐1KT) following the manufacturer's instructions.