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25978
CD57 (HNK-1) Mouse mAb (Pacific Blue™ Conjugate)
Antibody Conjugates
Monoclonal Antibody

CD57 (HNK-1) Mouse mAb (Pacific Blue Conjugate) #25978

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Flow cytometric analysis of human peripheral blood mononuclear cells gated on lymphocytes co-stained with NCAM1 (CD56) (MY31) Mouse mAb (APC Conjugate) #51997 using CD57 (HNK-1) Mouse mAb (Pacific Blue Conjugate) (right) compared to a concentration-matched Mouse IgM Isotype Control (Pacific Blue Conjugate) (left).
To Purchase # 25978
Cat. # Size Qty. Price
25978S
200 µl  (100 tests)

Supporting Data

REACTIVITY H
SENSITIVITY Endogenous
MW (kDa)
Source/Isotype Mouse IgM

Application Key:

  • WB-Western Blot
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • C&R-CUT&RUN
  • C&T-CUT&Tag
  • DB-Dot Blot
  • eCLIP-eCLIP
  • IF-Immunofluorescence
  • F-Flow Cytometry

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Vir-Virus
  • 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
  • GP-Guinea Pig
  • Rab-Rabbit
  • All-All Species Expected

Product Description

This Cell Signaling Technology antibody is conjugated to Pacific Blue fluorescent dye and tested in-house for direct flow cytometric analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated antibody CD57 (HNK-1) Mouse mAb #72031.

Product Usage Information

Application Dilution
Flow Cytometry (Fixed/Permeabilized) 1:50
Flow Cytometry (Live) 1:50

Storage

Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. This product is stable for 12 months when stored at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.

Protocol

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Flow Cytometry Triton™ X-100 Permeabilization Protocol for Mouse Antibodies

A. Solutions and Reagents

All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Triton™ X-100) #51995, 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. Cell Permeabilization Buffer: Purchase ready-to-use (#39487) or to prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml Antibody Dilution Buffer. Store at 4°C.
  4. Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616), or to prepare 100 ml dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
  5. Recommended Anti-Mouse secondary antibodies:
    • Anti-Mouse IgG (H+L), F(ab′)2 Fragment (Alexa® 488 Conjugate) #4408
    • Anti-Mouse IgG (H+L), F(ab′)2 Fragment (Alexa® 594 Conjugate) #8890
    • Anti-Mouse IgG (H+L), F(ab′)2 Fragment (Alexa® 647 Conjugate) #4410
    • Anti-Mouse IgG (H+L), F(ab′)2 Fragment (PE Conjugate) #59997

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. Fixation and Permeabilization

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 Triton™ X-100. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. 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.
  5. Resuspend cells in approximately 100 µl Cell Permeabilization Buffer per million cells.
  6. Incubate for 10 minutes at room temperature.
  7. Proceed with staining or store cells at 4°C in PBS overnight.

C. 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. Centrifuge cells and discard 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 1 hr at room temperature (20-25°C).
  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 recommended dilution).
  7. Incubate for 30 minutes at room temperature (20-25°C). Protect from light.
  8. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
  9. Resuspend cells in 1X PBS and analyze on flow cytometer.

posted January 2017

revised June 2020

Protocol Id: 1345

Flow Cytometry, Live Cell Protocol for Directly Conjugated 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.

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: Human Fc receptors cross-react with rabbit IgG. When cells of interest express high levels of Fc receptor protein (for example, macrophage/monocyte lineages), pre-incubate live cells with human Fc block prior to immunostaining with rabbit antibodies.

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. Discard supernatant. Repeat.
  6. Resuspend cells in 200-500 µl of Antibody Dilution Buffer and analyze on flow cytometer.

posted June 2017

revised January 2022

Protocol Id: 1504

Specificity / Sensitivity

CD57 (HNK-1) Mouse mAb (Pacific Blue Conjugate) recognizes the CD57 carbohydrate modification produced by the B3GAT1 protein enzyme.

Species Reactivity:

Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a membrane antigen from HSB-2 cells.

Background

CD57 antigen, also known as HNK-1 and Leu7, is a terminally sulfated glycan carbohydrate epitope (glyco-epitope) expressed on a variety of proteins, lipids, and chondroitin sulfate proteoglycans at the cell surface (1,2). CD57 is synthesized by the enzyme B3GAT1 and is present on a subset of peripheral blood lymphocytes, including NK cells and CD8+ T cells, as well as neural cells and striated muscle (3-5). Studies have shown that CD57 is not expressed on platelets, monocytes, granulocytes, or red blood cells (6). The CD57 epitope may play a role in neural cell adhesion (7), and characterizes unique maturation states in T and NK cells (8,9). CD57 is an important marker when studying functional immune deficiency in patients with autoimmune diseases, infectious diseases, and cancers (1).

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

For Research Use Only. Not for Use in Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Pacific Blue is a trademark of Molecular Probes, Inc.
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