Alexa fluor 594 anti mouse cd19 antibody

Lymph node cells isolated from C57BL/6 J female mice were stained with Alexa Fluor® 594 anti-mouse CD19 Antibody (cat# 115552, BioLegend) and Alexa Fluor® 488 anti-mouse CD3 Antibody (cat# 100210, BioLegend) for 30 min at 4 °C. Following the staining process, the cell suspension was placed into glass bottomed dishes. Once the cells settled naturally to the dish’s bottom surface, images were captured utilizing the Leica TCS SP5 II laser confocal microscopy system.

For inhibition of formin and Arp2/3, cells were incubated with inhibitors for 5 min at 37 °C before being added to the imaging chamber, which had the inhibitor at the same concentration used for incubation. SMIFH2 (Sigma-Aldrich) was used at a 25 μM concentration. Arp2/3 complex inhibitor I, CK666 (Calbiochem) was used at 50 μM. For N-WASP inhibition, wiskostatin B (EMD Bioscience) was used at 10 μM to incubate cells for 1 h at 37 °C. Mono-biotinylated fragment of antibody (mbFab′-anti-Ig) was generated from the F(ab′)₂ fragment (Jackson Immuno Research, West Grove PA) using a published protocol^{51 (link)}. FcγRIIB (CD32) antibody (Cat# 553141, BD Biosciences) was conjugated with Alexa Fluor 546 using Molecular Probes Protein labeling kits (Cat# A10237, Invitrogen) following manufacturer protocols. For labeling of CD19 we used the Alexa Fluor 594 anti-mouse CD19 antibody at 0.15 μg/ml (Cat# 115552, BioLegend).