Confocal microscopy and total internal reflection fluorescence microscopy
To observe the surface distribution of B cell receptors (BCR) and their related physiological activities, confocal microscopy (CFm) and total internal reflection fluorescence microscopy (TIRFm) were utilized. For CFm assay, purified B cells were incubated with AF594-mB-F(ab’)2-anti-Ig (M + G) (115-586-068; Jackson ImmunoResearch) and activated at 37 °C for 5, 10, and 30 min. The cells were fix and permeabilize before staining. The Abs used were purchased from Cell Signaling Technology (Danvers, MA, USA): pSHIP (3941S), pSTAT1 (9167S), pSTAT5 (4322S), and pNF-κB (3033S). From Abcam (Cambridge, MA, USA): pBtk (ab52192), pCD19 (ab203615). From Thermo Fisher: F-actin (R37110), AF488 G/R IgG (A-11008), AF405 G/R IgG, and AF405 G/M IgG (A-31553). From Bethyl Laboratories: pWASP (A300-205A). From Merck-Millipore (Darmstadt, Germany): pY (05-321).
For TIRFm assay, B cells were stimulated with membrane-tethered antigens (mAg) using lipid bilayers consisting of biotinylated F(ab’)2 and streptavidin tethered to lipids attached to the bottom of chambers. As previously described,23cells were labeled with AF546-Fab’ before being stimulated with mAg for 3, 5, and 7 min. Following stimulation, the cells were fixed, permeabilized, and stained with the same Abs used in the CFm assays.
Representative images were taken and analyzed for mean fluorescence intensity (MFI), area of B cell spreading (IRM), and colocalization using NIS elements AR 5.01 software (Nikon Eclipse Ti-PFS, Tokyo, Japan). Data from three independent experiments using more than 30-50 individual cells were included for each parameter. In order to minimize the variability, a unified evaluation standard to evaluate the morphology and contact area at different time points was set in all our studies.