FITC Mouse Anti-Human CD22

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Live cells were identified using LIVE/DEAD Fixable Near-IR staining (Molecular Probes) according to the manufacturer’s instructions. Cultured cells were pelleted, washed, and stained with LIVE/DEAD, followed by surface staining with fluorescently labeled mAbs	Multicolor flow cytometry was performed using a five-laser LSRII flow cytometer (BD) and analyzed with FlowJo software (Tree Star).

Protocol tips
Live cells were identified using LIVE/DEAD Fixable Near-IR staining (Molecular Probes) according to the manufacturer’s instructions. Cultured cells were pelleted, washed, and stained with LIVE/DEAD, followed by surface staining with fluorescently labeled mAbs
Downstream tips
Multicolor flow cytometry was performed using a five-laser LSRII flow cytometer (BD) and analyzed with FlowJo software (Tree Star).

Publication protocol

For characterization of tonsillar B-cell subsets, single cell suspensions were stained with appropriate combinations of fluorescently labeled mAbs, including anti-CD10 (CB-CALLA), CD19 (SJ25C1), CD20 (2H7), CD22 (4KB128 and S-HCL-1), CD27 (LG.7F9), CD38 (HB7) (eBioscience), IgD (IA6-2), CD3 (SP34-2), and CD95 (DX2) (BD Biosciences). Live cells were identified using LIVE/DEAD Fixable Near-IR staining (Molecular Probes) according to the manufacturer’s instructions. Cultured cells were pelleted, washed, and stained with LIVE/DEAD, followed by surface staining with fluorescently labeled mAbs. For Blimp1 intracellular staining, cells were first stained with LIVE/DEAD fixable dye, washed, stained with appropriate surface markers, washed and then fixed, permeabilized, and stained with PE-conjugated rat IgG2ak anti-Blimp1 Ab (6D3) using the Transcription Factor Buffer Set (BD). CFSE-labeled cells were cultured for 3 days and the levels of cell proliferation were measured based on CFSE dilution. Multicolor flow cytometry was performed using a five-laser LSRII flow cytometer (BD) and analyzed with FlowJo software (Tree Star).

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