Anti b220 percp

Apoptosis was measured by staining with annexin V FITC (BD Biosciences).
To assay Igλ usage, splenocytes were depleted of red blood cells and
stained with anti-CD21-FITC, anti-CD23-PE, anti-B220-PerCP and
anti-Igλ_1,2,3-biotin (BD Biosciences or Tonbo
Biosciences). The biotinylated antibody was detected with streptavidin-
allophycocyanin (APC) (BD Biosciences). Bone marrow cells were depleted of red
blood cells and stained with anti-B220 FITC,
anti-Igλ_1,2,3-biotin, anti-B220-PerCP, and anti-CD93/AA4.1-APC
(BD Biosciences or Tonbo Biosciences). The biotinylated antibody was detected
with streptavidin-PE (BD Biosciences). T3 cells were identified by staining
splenocytes with anti-B220 FITC, anti-CD23-PE, anti IgM-PerCP, and anti-AA4.1
APC (BD Biosciences or Tonbo Biosciences). In studies with the MD4 anti-HEL Ig
transgene system, splenocytes were depleted of red blood cells and stained with
combinations of anti-IgMb-FITC, anti-IgMa-PE, anti-B220 PerCP, anti-CD19 APC
(antibodies from BD Biosciences or Tonbo Biosciences), and hen egg lysozyme
(Sigma) labeled with Alexa 488 using an Alexa Fluro 488 Microscale Protein
Labeling Kit (Molecular Probes) according to the manufacturer’s
instructions.
Samples were run on a FACS Calibur (Becton Dickinson). Data were analyzed
with CellQuest (BD Biosciences) or Flowjo (Treestar).

A20 cell line, derived from mouse B cell lymphoma on BALB/c background [13 (link)], was obtained from American Type Culture Collection (Manassas, VA). Primary cells were isolated from bone marrow (BM), spleen (SPL) or peripheral blood (PB) of B6 by standard methods. The A20 transfectants or primary cells were stained with the following antibodies (Abs): anti-mouse CEACAM1 mAb, CC1 (kindly provided by Dr. Kathlyn Holmes, University of Colorado, CO), anti-IgG1, FITC-anti-B220, PE-anti-IgM, FITC-anti-CD4, PE-anti-CD3ε, PerCP-anti-B220, PE-anti-CD43, PE-anti-CD25, PE-anti-Igκ, PE-anti-CD138, PE-anti-CD5, PE-anti-H-2K^d, PE-anti-I-A^d, PE-anti-CD69, PE-anti-CD80 and PE-anti-CD86 Abs (BD Biosciences, San Jose, CA). Data acquisition was performed using FACS Calibur^™ and analysis software Cell Quest^™ (BD Biosciences).
In some experiments, the A20 transfectants were treated with Fluo-4® (Dojindo, Kumamoto, Japan), and applied for FACS to analyze intracellular Ca²⁺ influx. During the acquisition, cells were treated with anti-Igκ (Southern Biotech, Birmingham, AL) to stimulate BCR signaling, and kinetics for Ca²⁺ influx were measured at FL2.