Anti igm apc

Carboxylate modified latex (CML) beads with the diameter of 10 µm (Invitrogen) were coupled with NIP(15)BSA-biotin (Biosearch) following manufacturer's instruction. Briefly, 250 µl of CML beads (40 mg/ml) were first washed twice with 1 ml MES buffer (25 mM, pH6) and then resuspended in 500 µl MES. 200 µl of freshly prepared EDAC (1 Ethyl 3-(3-Dimethyl Amino Propyl) Carbodiimide HCl, Sigma-Aldrich, 50 mg/ml in MES) and 300 µl NIP(15)BSA-biotin (Biosearch, 6.6 μg/ml in MES) were added to the latex suspension and incubated at room temperature with gentle mixing for 4 hr. The resulting beads were then washed three times with 1 ml PBS and resuspended in 1 ml storage buffer (1X PBS with 0.1% glycine and 0.1% NaN3).
Purified monomeric and pentameric IgM were loaded on beads by incubating 100,000 beads with 0.05 µM (monomeric) or 0.01 µM (pentameric) of IgM in 30 µl volume at room temperature for 30 min under mild shaking. The equal loading of the monomeric and pentameric IgM was verified by staining the beads with anti-IgM-APC (eBioscience) antibody and monitoring the staining by FACScan. After loading, the beads were washed with PBS and PLA were performed as described. The PLA signal was quantified by a FACScan on LSRFortessa (Becton Dickinson, Franklin Lakes, NJ) using the filter set for PE. Data were exported and analyzed and plotted using FlowJo software (TreeStar, Ashland, OR).

Bone marrow cells from femur and tibia bones were flushed out with PBS using a 25 G syringe needle. Spleens were disrupted and pulp dispersed in PBS. Erythrocytes were lysed with RBC lysis solution (Biological industries) and cells were washed. When indicated, cells were isolated on magnetic MACS columns (Miltenyi Biotech) by positive selection with either αCD19 magnetic beads or streptavidin magnetic beads and biotinylated αB220 (Miltenyi Biotech), according to the manufacturer’s instructions. Cell purity following isolation was assayed as <95% by flow cytometry (LSR II, BD Bioscience).
Cells from erythrocyte disrupted spleens and bone marrows were stained with the antibodies indicated and cellular composition was analyzed by flow cytometry (LSR II, BD Bioscience). The antibodies used in this report include anti-mouse-Igκ-PE (Southern Biotech), anti-human-Igκ-FITC (Southern Biotech), anti-IgM-APC (eBioscience), anti-B220-PerCP-Cy5.5 (Biolegend), anti-CD43-PE (Biolegend), anti-IgD-FITC (eBioscience). Flow cytometry output was analyzed using Flowing Software v2.5.0 (Turku Centre for Biotechnology).