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60040
KIR2DL3 (D8L3D) Rabbit mAb
Primary Antibodies
Monoclonal Antibody

KIR2DL3 (D8L3D) Rabbit mAb #60040

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Flow Cytometry Image 1: KIR2DL3 (D8L3D) Rabbit mAb

Flow cytometric analysis of live human peripheral blood mononuclear cells using KIR2DL3 (D8L3D) Rabbit mAb compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900. Samples were co-stained with CD56 to distinguish NK cell population. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412. Total viable cells were used for analysis.

To Purchase # 60040S
Product # Size Price
60040S
100 µl $ 268

Supporting Data

REACTIVITY H
SENSITIVITY Endogenous
MW (kDa) 60
Source/Isotype Rabbit IgG

Application Key:

  • W-Western
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • IF-Immunofluorescence
  • F-Flow Cytometry
  • E-P-ELISA-Peptide

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • All-All Species Expected

Product Usage Information

Application Dilution
Flow Cytometry 1:100

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Protocol

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Flow Cytometry, Live Cell Protocol for Unconjugated Rabbit Antibodies

A. Solutions and Reagents

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. 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.
  3. Recommended Anti-Rabbit secondary antibodies::
    • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412
    • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 594 Conjugate) #8889
    • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414
    • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (PE Conjugate) #79408

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 for a full listing of cellular dyes validated for use in flow cytometry.

B. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

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

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.

  1. Aliquot desired number of cells into tubes or wells. (Generally, 5x105 to 1x106 cells per assay.)
  2. Pellet cells by centrifugation and remove supernatant.
  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 30 min to 1 hr on ice. Protect from light.
  5. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
  6. Resuspend cells in 100 µl of diluted fluorochrome-conjugated secondary antibody (prepared in Antibody Dilution Buffer at the recommended dilution).
  7. Incubate for 30 min on ice. Protect from light.
  8. Wash by centrifugation in Antibody Dilution Buffer. Discard supernatant. Repeat.
  9. Resuspend cells in 200-500 µl of Antibody Dilution Buffer and analyze on flow cytometer.

revised June 2020

Protocol Id: 1865

Specificity / Sensitivity

KIR2DL3 (D8L3D) Rabbit mAb recognizes endogenous levels of total KIR2DL3 protein. This antibody weakly cross-reacts with KIR2DL2 proteins in over-expression cell lines.

Species Reactivity:

Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala173 of human KIR2DL3 protein.

Background

Killer cell immunoglobulin-like receptors (KIRs) are type 1 transmembrane glycoproteins expressed by natural killer cells and subsets of CD4, CD8, and γδ T cells (1-5). Analogous to the diversity of their human leucocyte antigen class I (HLA Class I) ligands, the KIR genes are polymorphic and the content of the KIR gene cluster varies among haplotypes, although several "framework" genes are found in all haplotypes (6-7). The KIR proteins are characterized by the number of extracellular immunoglobulin-superfamily domains (2D or 3D) and by whether they have a long (L) or short (S) cytoplasmic domain (8-10). KIR proteins with the long cytoplasmic domain transduce inhibitory signals upon ligand binding via an immune tyrosine-based inhibitory motif (ITIM) (10), while KIR proteins with the short cytoplasmic domain lack an ITIM and instead transduce activating signals (11,12). KIR proteins play an important role in the regulation of the immune response. Combinations of KIR and HLA class I variants influence susceptibility to autoimmunity and infectious disease, as well as outcomes of haematopoietic stem cell transplantation (12-14).

KIR2DL3, also referred to as CD158b, interacts with HLA-C alleles (HLA-Cw1, HLA-Cw3, and HLA-Cw7). Upon receptor ligand interaction, KIR2DL3 inhibits the activity of NK cells thus preventing target cell lysis (15-17).

  1. Young, N.T. et al. (2001) J Immunol 166, 3933-41.
  2. Battistini, L. et al. (1997) J Immunol 159, 3723-30.
  3. Björkström, N.K. et al. (2012) Blood 120, 3455-65.
  4. Remtoula, N. et al. (2008) J Immunol 180, 2767-71.
  5. Béziat, V. et al. (2017) Immunology 150, 248-264.
  6. Uhrberg, M. et al. (1997) Immunity 7, 753-63.
  7. Shilling, H.G. et al. (2002) J Immunol 168, 2307-15.
  8. Fan, Q.R. et al. (2001) Nat Immunol 2, 452-60.
  9. Boyington, J.C. et al. (2000) Nature 405, 537-43.
  10. Vivian, J.P. et al. (2011) Nature 479, 401-5.
  11. Stewart, C.A. et al. (2005) Proc Natl Acad Sci U S A 102, 13224-9.
  12. Ivarsson, M.A. et al. (2014) Front Immunol 5, 184.
  13. Kulkarni, S. et al. (2008) Semin Immunol 20, 343-52.
  14. Martin, M.P. and Carrington, M. (2013) Immunol Rev 254, 245-64.
  15. Colonna, M. et al. (1993) Proc Natl Acad Sci U S A 90, 12000-4.
  16. Winter, C.C. et al. (1998) J Immunol 161, 571-7.
  17. Moesta, A.K. et al. (2008) J Immunol 180, 3969-79.

Pathways & Proteins

Explore pathways + proteins related to this product.

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