Alexafluor 467 goat anti mouse igm

Cells were cultured in 4-well chamber slides (Nunc Lab-Tek II, Thermo Fischer) at a density of 5 × 10⁵ cells mL⁻¹. CD40L-beads and anti-IgM antigen mimic were added to the culture. At 0 and 30 min, the media was removed and the cells were fixed in Cytofix Fixation Buffer (BD Biosciences) for 20 min at room temperature. The samples were washed twice in PBS and stored overnight at 4 C. The following day, the samples were permeabilized in 0.1% Triton X in PBS (Sigma-Aldrich) and subsequently blocked in 0.5% BSA in PBS (Sigma-Aldrich) for 10 min each. BCRs were labeled with Alexafluor 467 goat-anti-mouse IgM (Abcam, Cambridge, UK). Samples were imaged on a Zeiss LSM 780 Confocal Microscope (Zeiss, Oberkochen, DE) and analyzed using ImageJ software (NIH, Bethesda, MD, USA). BCR cluster area was determined using a custom ImageJ macro to calculate the total area of pixels above an intensity threshold.