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26969
FcγRIIB (D8F9C) XP® Rabbit mAb (Mouse Specific) (PE Conjugate)

FcγRIIB (D8F9C) XP® Rabbit mAb (Mouse Specific) (PE Conjugate) #26969

APPLICATIONS

REACTIVITY SENSITIVITY MW (kDa) Isotype
M Endogenous Rabbit IgG
Flow Cytometry

Flow cytometric analysis of mouse splenocytes using FcγRIIB (D8F9C) XP® Rabbit mAb (Mouse Specific) (PE Conjugate) (right) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (left). Samples were co-stained with CD45R (B220)-APC to distinguish the B cell population.

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Flow Cytometry, Methanol Permeabilization Protocol for Direct Conjugates

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 16% Formaldehyde (methanol free).
  3. 100% methanol.
  4. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Fixation

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Resuspend cells in 0.5-1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
  3. Fix for 15 min at room temperature.
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS.

C. Permeabilization

  1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
  2. Incubate 30 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

  1. Aliquot desired number of cells into tubes or wells.
  2. Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
  3. Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
  4. Incubate for 1 hr at room temperature. Protect from light.
  5. Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
  6. Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).

E. Optional DNA Dye

  1. Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
  2. Incubate for at least 5 min at room temperature.
  3. Analyze cells in DNA staining solution on flow cytometer.

posted July 2009

revised June 2017

Protocol Id: 407

Application Dilutions
Flow Cytometry 1:50
Storage:

Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.

FcγRIIB (D8F9C) XP® Rabbit mAb (Mouse Specific) (PE Conjugate) recognizes endogenous levels of total FcγRIIB protein.

Species Reactivity:

Mouse

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro284 of mouse FcγRIIB protein.

This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometric analysis in mouse cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated FcγRIIB (D8F9C) XP® Rabbit mAb (Mouse Specific) #96397.

FcγRIIB (CD32B) is a low affinity, IgG Fc-binding receptor expressed on B cells, monocytes, macrophages, and dendritic cells (DCs) (1-3). It is the inhibitory Fc receptor and signals through an immunoreceptor tyrosine-based inhibitory motif (ITIM) within its carboxy-terminal cytoplasmic tail (2). Binding of immune complexes to FcγRIIB results in tyrosine phosphorylation of the ITIM motif at Tyr292 and recruitment of the phosphatase SHIP, which mediates inhibitory effects on immune cell activation (2,4). In this way, FcγRIIB suppresses the effects of activating Fc-binding receptors (3). For example, mice deficient for FcγRIIB have greater T cell and DC responses following injection of immune complexes (5, 6). In addition, FcγRIIB plays a role in B cell affinity maturation (7). Signaling through FcγRIIB in the absence of signaling through the B cell receptor (BCR) is proapoptotic, while signaling through FcγRIIB and the BCR simultaneously attenuates the apoptotic signal and results in selection of B cells with higher antigen affinity (7).

  1. Tridandapani, S. et al. (2002) J. Biol. Chem. 277, 5082-5089.
  2. Tridandapani, S. et al. (1997) Mol. Cell. Biol. 17, 4305-4311.
  3. Guilliams, M. et al. (2014) Nat Rev Immunol 14, 94-108.
  4. Bruhns, P. et al. (2000) J. Biol. Chem. 275, 37357-37364.
  5. Kalergis, A.M. and Ravetch, J.V. (2002) J Exp Med 195, 1653-9.
  6. Desai, D.D. et al. (2007) J Immunol 178, 6217-26.
  7. Pearse, R.N. et al. (1999) Immunity 10, 753-60.
Entrez-Gene Id
14130
Swiss-Prot Acc.
P08101
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.

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Product # Size Price
26969S
100 µl (50 tests) $ 348.0