Anti b220 fitc

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).

For CD4⁺CD25⁺Foxp3⁺ T cells, splenocytes were isolated using standard methods. Nuclear stain for transcription factor FoxP3 was performed with the BD Biosciences (San Jose, CA) kit. Briefly, splenocytes (1×10⁶ per mouse) were stained with fluorescent conjugated antibodies, including anti-mouse CD4-pacific blue, anti-mouse CD25-PE and anti-mouse FoxP3-Alexa Fluor 647 according to staining protocol. For TfH cell staining, anti-CXCR5-BV421, anti-CD4-BV605, anti-PD-1-APC, anti-B220-FITC, and anti-Bcl6-PE were purchased from BD Bioscience (San Jose, CA). Fixable viability dye was purchased from eBioscience (San Diego, CA). For dendritic cell characterization, antiCD11b, anti-CD11c, and anti-CD64 were purchased from eBioscience (San Diego, CA). FACS analysis using LSR-II instrumentation from BD Biosciences (San Jose, CA) and the FCS Express software (DeNovo Software).

Bone marrow was first depleted of red blood cells and B cells were
then purified with anti-B220 magnetic beads using the IMag system (BD
Pharmingen). B cells were subsequently stained with anti-IgM-PE,
anti-B220-PerCP or anti-B220-FITC, and anti-CD93/AA4.1-biotin or
anti-CD93/AA4.1-APC (BD Biosciences or Tonbo Biosciences). The biotinylated
antibody was detected with streptavidin-APC (Caltag or BD Biosciences).
Immature (B220+, IgM+, AA4.1+) and pre (B220+, IgM-, AA4.1+) B cells were
sorted on a FACS Aria (Becton Dickinson) or a MoFlo (Cytomation) cell
sorter. Samples were kept at 4° at all times prior to and during the
sort and until stimulation to avoid activating the BCR with the sorting
antibodies (11 (link)). Purified cells were
either harvested immediately or stimulated with 10 μg/ml goat
anti-mouse IgM F(ab’)₂ (Jackson ImmunoResearch
Laboratories) for the indicated times at 10⁶ cells/ml in RPMI +
10% FBS. Alternatively, bone marrow B lineage cells were expanded by
culturing for 4–6 days in 10 ng/ml IL-7 (R & D Systems) at 2
× 10⁶ cells/ml and pre and immature B cells sorted as
above.