Multicolor Flow Cytometry of Splenocytes

Single cell suspensions of splenocytes were treated with ACK lysis buffer (155 mM NH₄Cl, 10 mM KHCO₃, 100 μM EDTA) to remove red blood cells before staining in PBS, containing LIVE/DEAD fixable near-IR dead cell stain (Life Technologies) and appropriate, pre-titered antibodies. Antibodies used, indicating antigen and fluorophore (and clone): CD69-FITC (H1.2F3), CD86-PE (GL-1), Streptavidin-PerCP (Becton Dickinson), MHC class II-bio (I-Ab) (M5/114.15.2), IgM-PECy7 (II/41), B220-eFluor450 (RA3-6B2) (eBiosciences), CD19-FITC, B220-PerC, IgM-PE, IgG1-APC, PNA-FITC, CD38-PE/Cy7, CD45.2-ef450, CD45.1-FITC, CD138-PE, IgD-ef450, CD4-FITC, CD8-FITC, CD45.2-PerCP-cy5.5, CD45.1-FITC, B220-BV605, CD4-BV650, CD8a-BV650, CD38-bio, Streptavidin-PacO, FAS-PE. Anti-Syk Antibody (5F5) (Biolegend) and NIP-BSA were labelled using LYNX Rapid APC, RPE and PerCP Antibody Conjugation Kits (AbD Serotec). Antibody staining of intracellular Syk was carried out as previously described (13 (link)).

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Ackermann J.A., Nys J., Schweighoffer E., McCleary S., Smithers N, & Tybulewicz V.L. (2015). Syk Tyrosine Kinase Is Critical for B Cell Antibody Responses and Memory B Cell Survival. The Journal of Immunology Author Choice, 194(10), 4650-4656.

Publication 2015

LIVE/DEAD fixable near-IR dead cell stain CD69-FITC CD86-PE Streptavidin-PerCP B220-eFluor450 (RA3-6B2) CD19-FITC B220-PerC CD138-PE CD4-FITC CD45.2-PerCP Streptavidin-PacO

Anti antibody Antibodies Antibody Antigen Buffer Cd138 Cell Clone Cy5 5 Edta Fitc Igg1 Intracellular Khco3 Lynx Mhc class ii Red blood cells Stain Streptavidin

Corresponding Organization :

Other organizations : GlaxoSmithKline (United Kingdom)

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Variable analysis

independent variables

Treatment with ACK lysis buffer
Staining with pre-titered antibodies

dependent variables

Expression of CD69, CD86, MHC class II, IgM, B220, CD19, IgG1, PNA, CD38, CD45.2, CD45.1, CD138, IgD, CD4, CD8, Syk

control variables

Cell suspension preparation in PBS
Use of LIVE/DEAD fixable near-IR dead cell stain

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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