Cd57 fitc

Manufactured by Thermo Fisher Scientific

Sourced in United States

CD57-FITC is a fluorescently-labeled monoclonal antibody that binds to the CD57 cell surface antigen, also known as HNK-1 or Leu-7. CD57 is expressed on a subset of T cells and natural killer cells, and is often used as a marker for these cell types in flow cytometry and other immunological applications.

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Close spelling variants of this product

Anti-CD57-FITC (8 citations) FITC-conjugated anti-human CD57 (3 citations)

7 protocols using cd57 fitc

Immune Cell Phenotyping by Flow Cytometry

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0.5 × 10⁶ isolated PBMCs were stained with anti-CD3-eFluor450, CD4-APC-eFluor780, CD8-PerCP-CY5.5, CD27-APC, CD45RO-PE, CD57-FITC, CD38-PE-Cy7 (eBioscience) to distinguish subsets. Appropriate isotype controls were used to determine the percentages of cells expressing the respective markers (Biolegend, San Diego, CA). Samples were analyzed on LSR-II flow cytometer (BD) and data analysis was performed using the FACSDiva software (BD).

Michel K.G., Huijbregts R.P., Gleason J.L., Richter H.E, & Hel Z. (2015). Effect of hormonal contraception on the function of plasmacytoid dendritic cells and distribution of immune cell populations in the female reproductive tract. Journal of acquired immune deficiency syndromes (1999), 68(5), 511-518.

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Comprehensive Immunophenotyping of PBMCs

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0.5 x 10⁶ isolated PBMCs were stained with anti-CD3-PacBlue, CD4-APC-Alexa Fluor750, CD8-PerCP-CY5.5, CD27-APC, CD45RO-PE, CD57-FITC, and CD38-PE-Cy7 (eBioscience) to distinguish T cell subsets and levels of activation. B cell subsets and activation status was analyzed by staining with CD70-FITC, α-IgD-PE (BD), CD19-PE-Cy7, and CD27-APC (BD) antibodies. Appropriate isotype controls were used to determine the percentages of cells expressing the respective markers (Biolegend, San Diego, CA). Samples were analyzed on LSR-II flow cytometer (BD) and data analysis was performed using the FlowJo (FlowJo, LLC) and FACSDiva software (BD) as described [52 (link), 54 (link), 55 (link)].

Hel Z., Xu J., Denning W.L., Helton E.S., Huijbregts R.P., Heath S.L., Overton E.T., Christmann B.S., Elson C.O., Goepfert P.A, & Mestecky J. (2017). Dysregulation of Systemic and Mucosal Humoral Responses to Microbial and Food Antigens as a Factor Contributing to Microbial Translocation and Chronic Inflammation in HIV-1 Infection. PLoS Pathogens, 13(1), e1006087.

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Multiparametric Flow Cytometry Analysis of PBMCs

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PBMCs were thawed, stained with 5 μl of the HLA-A*02:01 dextramer conjugated to PE for 10 minutes at room temperature, followed by the addition of 100 μl of a mastermix of anti-membrane marker mAbs containing LIVE/DEAD fixable aqua stain (Molecular Probes, Invitrogen), CD3 ECD (Beckman Coulter), CD4 BV605 and CCR7 Pacific blue (Biolegend), CD8 Alexa Fluor 700, CD14 PE-Cy7, CD16 PE-Cy7, CD19 PE-Cy7, CD45RA APC, CD57 FITC, TIGIT PerCP-eFluor710, PD-1 APC-eFluor780 (eBiosciences) and CD27 Qdot 655 (Life Technologies). The cells were incubated at 4°C for a further 20 minutes, washed and fixed with 1% paraformaldehyde in PBS prior to running on an LSRII flow cytometer (Becton-Dickinson).

Moyo N., Borthwick N.J., Wee E.G., Capucci S., Crook A., Dorrell L, & Hanke T. (2017). Long-term follow up of human T-cell responses to conserved HIV-1 regions elicited by DNA/simian adenovirus/MVA vaccine regimens. PLoS ONE, 12(7), e0181382.

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Immunophenotyping of NK and CD8+ T Cells

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To identify NK and CD8⁺ T cells subsets, Blood samples (about 10 ml) were
collected in EDTA-containing tubes. The peripheral blood mononuclear cells (PBMCs) were
first isolated using Ficoll-Hypaque (Innotrain, Germany) density gradient centrifugation
(1000×g at room temperature) and then washed two times with phosphate-buffered saline
(PBS, Euroimmun, Germany). Centrifugation of samples was at 300 ×g and 4°C. Subsequently,
isolated PBMCs (10⁶ cells/100 µl) were stained with anti-CD8-APC, CD45RO-PE,
CD27-Percp-eFlour780, CD28-Pe-Cy7, CD57-FITC, and CD56-PerCp-eFlour710 monoclonal
antibodies (mAbs) (all from eBioscience, USA) and incubated at 4°C for 30 minutes. The
samples were fixed with formaldehyde and analyzed within 24 hours by flow cytometer (BD
FACSAria, USA). At least 50,000 events were counted for each sample.

Salehi Z., Beheshti M., Nomanpour B., Khosravani P., Naseri M., Sahraian M.A, & Izad M. (2021). The Association of EBV and HHV-6 Viral Load with Different NK and CD8+ T Cell Subsets in The Acute Phase of Relapsing-Remitting Multiple Sclerosis. Cell Journal (Yakhteh), 23(6), 626-632.

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Multicolor Flow Cytometric Analysis of Immune Cell Activation

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After 18 h incubation with IL-2 alone, IL-2 with LPS, or IL-2 with IL-12 (10 ng/ml; Sigma-Aldrich), immunofluorescent staining was performed using CD56-APC, CD57-FITC, and CD69-PE antibodies (eBioscience) followed by multicolor flow cytometric analysis.

Kanevskiy L.M., Erokhina S.A., Streltsova M.A., Telford W.G., Sapozhnikov A.M, & Kovalenko E.I. (2014). Bacterial Lipopolysaccharide Activates CD57-Negative Human NK Cells. Biochemistry. Biokhimiia, 79(12), 1339-1348.

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Comprehensive Immune Phenotyping of PBMCs

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PBMCs were thawed, incubated with 5 μl of the HLA-A*02:01/YQYMDDLV tetramer (NIH Tetramer Core Facility) conjugated to PE for 10 min at room temperature, followed by the addition of 100 μl of a mastermix of anti-membrane marker mAbs containing LIVE/DEAD fixable aqua stain (Molecular Probes, Invitrogen), CD3 ECD (Beckman Coulter), CD4 BV605 and CCR7 Pacific blue (Biolegend), CD8 Alexa Fluor 700, CD14 PE-Cy7, CD16 PE-Cy7, CD19 PE-Cy7, CD45RA APC, CD57 FITC, TIGIT PerCP-eFluor710, PD-1 APC-eFluor780 (eBiosciences) and CD27 Qdot 655 (Life Technologies). The cells were incubated at 4°C for a further 20 min, washed and fixed with 1% paraformaldehyde in PBS prior to running on an LSRII flow cytometer (Becton-Dickinson).

Borthwick N.J., Lane T., Moyo N., Crook A., Shim J.M., Baines I., Wee E.G., Hawkins P.N., Gillmore J.D., Hanke T, & Pepys M.B. (2018). Randomized phase I trial HIV-CORE 003: Depletion of serum amyloid P component and immunogenicity of DNA vaccination against HIV-1. PLoS ONE, 13(5), e0197299.

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NK Cell Isolation and Activation

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Blood samples were taken from healthy adult volunteers who have given informed consent for their blood to be used in this study. Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation on Ficoll density gradient 1.077 g/cm³ (Paneco, Russia). The NK cells were then magnetically separated using a human NK cell negative selection kit (Miltenyi Biotec, Germany). The percentage of CD3⁻CD56⁺ cells in the preparations after separation was no less than 97% as verified by flow cytometry. In some cases, NK subpopulations were isolated from magnetically separated NK cells by fluorescent-activated cell sorting after staining with monoclonal antibodies CD3-PC7, CD56-APC (Beckman Coulter, USA), and CD57-FITC (eBioscience, USA). Isolated NK cells were cultivated in RPMI-1640 (Paneco) supplemented with 10% FCS (HyClone, USA) (200 μl) at cell concentration of 1.5·10⁶ cells/ml or 0.5·10⁶ cells/ml (for sorted cells) for 18 h in 96 U-well plates (Costar, USA). Recombinant human IL-2 (500 U/ml) purchased from Hoffmann-La-Roche (Germany) and LPS (5 μg/ml) from E. coli strain 055:B5 (Sigma-Aldrich, USA) were added to the cell culture. After incubation, TLR4 and CD69 expression and cytotoxicity of NK cells were analyzed; cell-free supernatants were collected for estimation of cytokine production.

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