Fitc conjugated hamster anti rat cd29

CD29-ve RBL-2H3 cells were generated in the same manner as that described for the FcεRI-ve RBL-2H3 cell line but using the guide sequence 5’-TCATCACATCGTGCAGAAGT-3’, which targets nucleotides 178 to 197 of rat Itgb1. This sequence has a fidelity score of 75 (crispr.mit.edu). A pure population of cells was sorted by staining with FITC-conjugated hamster anti-rat CD29 (Biolegend; 102205).

In all cases, cells were labeled with antibody/protein at 1 μg/ml in PBS + 0.05% sodium azide for 1 hour at 4°C. Cells were washed with three times with 4 ml of cold PBS-azide before resuspension in 400 μl of PBS-azide. Fluorescence was measured with a CyAn ADP flow cytometer (Beckman Coulter). To assess cell surface integrin expression, FITC-conjugated hamster anti-rat CD29 (Biolegend; 102205) and FITC-conjugated mouse anti-rat CD49d (Biolegend; 200103) were used. For general rFcεRI staining, mouse anti-TNP IgE conjugated to Alexa Fluor 647 was used. Primary human basophils were stained with anti-CD123 FITC (Biolegend; 306005) and anti-human IgE APC (Biolegend; 325507). To assess Fcε construct binding, RBL-2H3 cells were stained with the relevant Alexa Fluor 647–labeled protein, washed, and then incubated at 37°C in 4 ml of PBS-azide for various times after washing to enable dissociation of Fcε. Cells were then pelleted and resuspended in 400 μl of PBS-azide before analysis.