Biotinylated anti igm 315 065 049

Single cell suspensions of splenocytes, bone marrow, and mesenteric lymph nodes were prepared from fresh tissue with Ficoll purification. Cells were counted and added to the plate (3654-WP-10 ManTech) previously coated with 10 μg/mL anti-IgG (315-005-046, JacksonImmuno Research Laboratories) or 10 μg/mL anti-IgM (315-005-049, JacksonImmuno Research Laboratories) in bicarbonate coating buffer, washed, and then blocked. Cells were incubated overnight at 37°C in a 5% CO₂ incubator. The following day cells were removed and the plate was washed 5 times with PBS. Then 0.1 μg/mL biotinylated anti-IgG (315-065-046, JacksonImmuno Research Laboratories) or 0.1 μg/mL biotinylated anti-IgM (315-065-049, JacksonImmuno Research Laboratories) in PBS/0.1% Tween20 was added at 100 μL per well and incubated for 2 hours at RT. After washing, ExtrAvidin-ALP (1:1000) (Sigma Aldrich, E2636) in PBS was added at 100 μL per well, incubated 1 hour at RT. After washing, 100 μL per well of substrate (3650–10 BCIP/NBT-plus MabTech) was added and allowed to develop until distinct spots emerged. Color development was stopped by washing extensively in tap water. After drying, spots were counted by hand using a dissecting microscope.

Single cell suspensions of splenocytes, bone marrow, mesenteric lymph nodes were prepared from fresh tissue with Ficoll purification. Cells were counted added to the plate (3654-WP-10 ManTech) previously coated with 10 μg/mL anti-IgG (315-005-046, JacksonImmuno Research Laboratories) or 10 μg/mL anti-IgM (315-005-049, JacksonImmuno Research Laboratories) in bicarbonate coating buffer, washed, then blocked. Cells were incubated overnight at 37°C in a 5% CO₂ incubator. The following day cells were removed the plate was washed 5 times with PBS. Then 0.1 μg/mL biotinylated anti-IgG (315-065-046, JacksonImmuno Research Laboratories) or 0.1 μg/mL biotinylated anti-IgM (315-065-049, JacksonImmuno Research Laboratories) in PBS/0.1% Tween20 was added at 100 μL per well incubated for 2 hours at RT. After washing, ExtrAvidin-ALP (1:1000) (Sigma Aldrich, E2636) in PBS was added at 100 μL per well, incubated 1 hour at RT. After washing, 100 μL per well of substrate (3650–10 BCIP/NBT-plus MabTech) was added allowed to develop until distinct spots emerged. Color development was stopped by washing extensively in tap water. After drying, spots were counted by h using a dissecting microscope.