Pe mouse antihuman cd29 antibody

Purified neutrophils were suspended in 1% BSA in HBSS at the concentration of 10⁶ cells/mL (100 μL/sample) and incubated with FITC-labeled anti-α₄ integrin antibody (5 μL/sample, FITC Mouse anti-human CD49d, BD Pharmingen) or FITC-labeled anti-β₂ integrin antibody (15 μL/sample, FITC Mouse anti-human CD18, BD Pharmingen) for 45 min at 4°C, as previously described [33 (link)]. After two washes with 1% BSA in HBSS, cells were resuspended in PBS and analyzed in a Guava EasyCyte Flow Cytometer (Millipore) and 10000 cells/sample were analyzed. Data were normalized with the relative fluorescence for nonspecific binding evaluated by exposing the cells to an isotype control monoclonal antibody FITC mouse IgG (Becton Dickinson Italia) and set to 0.
In another set of experiments purified neutrophils were suspended in 1% BSA in HBSS at the concentration of 10⁶ cells/mL (100 μL/sample) and incubated with the conformational sensitive phycoerythrin (PE)-labeled HUTS-21 monoclonal antibody (20 μL/sample, PE mouse antihuman CD29 antibody, BD Pharmingen) for 45 min at room temperature. Neutrophils were washed twice with 1% BSA in HBSS, resuspended in PBS and analyzed at the flow cytometry. 10000 cells/sample were analyzed. Data were normalized to nonspecific binding relative fluorescence evaluated by exposing the cells to an isotype control mAb (monoclonal antibody) and set to 0.

MDA-MB-231 and their derivatives W2KO, miR-29–expressing, and ITGB1KO cells were detached by trypsinization and washed with FACS staining buffer (PBS with 5% BSA). Cells at a density of 5 × 10⁵/mL were stained with conjugated PE Mouse Anti-Human CD-29 antibody (BD Pharmingen, catalog no. 555443) for 30 minutes followed by washing and resuspending the cells in 250 μL FACS staining buffer. Anti-Integrin β1 Antibody, activated clone HUTS-4 (Sigma, catalog no. MAB2079Z) was used to detect the activation of ITGB1. As a positive control for integrin activation, 0.5 mmol/L MnCl₂ was used to treat the cells for 10 minutes at 37°C. Processed cells were then run through FACSAria and all the data were analyzed using FlowJo software. Data were corrected for any nonspecific signals and the resultant fluorescence intensities were plotted graphically.