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

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

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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.

To Purchase # 26969S
Product # Size Price
26969S
100 µl  (50 tests) $ 348

Supporting Data

REACTIVITY M
SENSITIVITY Endogenous
MW (kDa)
Isotype Rabbit IgG

Product Description

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.

Product Usage Information

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.

Protocol

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Flow Cytometry, Methanol Permeabilization Protocol for Directly Conjugated Antibodies

A. Solutions and Reagents

All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below.

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

  1. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100 ml 10X PBS (#12528) to 900 ml water mix.
  2. 4% Formaldehyde, Methanol-Free (#47746)
  3. 100% Methanol (#13604): Chill before use
  4. Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616), or prepare a 0.5% BSA PBS buffer by dissolving 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com/flowdyes for a listing of cellular dyes validated for use in flow cytometry.

B. Fixation

NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.

NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.

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

NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.

  1. Pellet cells by centrifugation and remove supernatant.
  2. Resuspend cells in approximately 100 µl 4% formaldehyde per 1 million cells. Mix well to dissociate pellet and prevent cross-linking of individual cells.
  3. Fix for 15 min at room temperature (20-25°C).
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS. Proceed to Permeabilization step.
    1. Alternatively, cells may be stored overnight at 4°C in 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. Permeabilize for a minimum of 10 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

  1. Aliquot desired number of cells into tubes or wells. (Generally, 5x105 to 1x106 cells per assay.)
  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 primary antibody, prepared in Antibody Dilution Buffer at a recommended dilution or as determined via titration.
  4. Incubate for 1 hr at room temperature. Protect from light.
  5. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
  6. Resuspend cells in 200-500 µl of 1X PBS and analyze on flow cytometer.

posted July 2009

revised August 2019

Protocol Id: 407

Specificity / Sensitivity

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

Species Reactivity:

Mouse

Source / Purification

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

Background

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, 5082-89.
  2. Tridandapani, S. et al. (1997) Mol Cell Biol 17, 4305-11.
  3. Guilliams, M. et al. (2014) Nat Rev Immunol 14, 94-108.
  4. Bruhns, P. et al. (2000) J. Biol. Chem. 275, 37357-64.
  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.

Pathways & Proteins

Explore pathways + proteins related to this product.

For Research Use Only. Not For Use In Diagnostic Procedures.

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