Anti cd8 amcyan

Manufactured by BD

Anti-CD8-AmCyan is a fluorescently labeled antibody that binds to the CD8 cell surface receptor. It is designed for use in flow cytometry applications to identify and enumerate CD8-positive cells.

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Lab products found in correlation

Anti cd3 apc cy7, by BioLegend (3 mentions) Anti ccr7 fitc, by R&D Systems (2 mentions) Anti pd 1 pe cy7, by BioLegend (1 mentions) Anti cd3 pacific blue, by BD (1 mentions) Anti cd8 v500, by BD (1 mentions) Anti cd27 apc efluor780, by Thermo Fisher Scientific (1 mentions) Anti ccr7 pe cy7, by BD (1 mentions) Anti ki67 fitc, by BD (1 mentions) 7 amino actinomycin d 7 aad via probe, by BD (1 mentions) Facscanto 2 flow cytometer, by BD (1 mentions) Anti cd160 pe, by BioLegend (1 mentions) Live dead blue dye, by Thermo Fisher Scientific (1 mentions) Anti pd 1 percpcy5, by BD (1 mentions) Anti ctla4 pe cy7, by BioLegend (1 mentions) Anti cd244 fitc, by BioLegend (1 mentions) Anti cd45ra af700, by BioLegend (1 mentions) Ifn γ af700, by BioLegend (1 mentions) Cd107a fitc, by BioLegend (1 mentions) Tnf α pe cy7, by Thermo Fisher Scientific (1 mentions) Cell stimulation cocktail, by Thermo Fisher Scientific (1 mentions)

5 protocols using anti cd8 amcyan

Phenotypic Characterization of Immune Cells

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The following monoclonal antibodies (MAbs) were used for phenotypic analysis: (i) anti-CD27-APC-eFluor780, anti-CD45RA-phycoerythrin (PE), and anti-CD127-eFluor450 (eBioscience, San Diego, CA); (ii) anti-CD3-Pacific Blue, anti-CD8-AmCyan, anti-CD8-V500, anti-CD11a-fluorescein isothiocyanate (FITC), anti-CD95-PE, anti-Ki67-FITC, and anti-CCR7-PE-Cy7 (BD Biosciences, Heidelberg, Germany); (iii) anti-CCR7-FITC (R&D Systems); and (iv) anti-PD-1-PE-Cy7 (BioLegend, San Diego, CA). 7-Aminoactinomycin D (7-AAD; Viaprobe; BD Biosciences) was used for the exclusion of dead cells. All samples were acquired using a FACSCanto II flow cytometer (BD Biosciences) and analyzed with FlowJo software (TreeStar Inc., Ashland, OR).

Nitschke K., Flecken T., Schmidt J., Gostick E., Marget M., Neumann-Haefelin C., Blum H.E., Price D.A, & Thimme R. (2014). Tetramer Enrichment Reveals the Presence of Phenotypically Diverse Hepatitis C Virus-Specific CD8+ T Cells in Chronic Infection. Journal of Virology, 89(1), 25-34.

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Detailed Immunophenotyping of T Cell Subsets

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Immunophenotyping was undertaken following APC-conjugated HLA class I tetramer staining of PBMCs at 37°C for 15 min. Details of the tetramers used can be found in Supplementary Table 5. Tetramers were conjugated to APC and a true tetramer response was verified through the lack of background staining by gating all CD3+ T cells, against CD8+ T cells and using a tetramer negative control. Surface staining with the following antibodies was then performed: live/dead blue dye (Invitrogen), anti-CD8 Amcyan (BD Biosciences), anti-CD3 APC-Cy7 (Biolegend), anti-PD-1 PercpCy5.5 (BD Biosciences), anti-CTLA4 PE-Cy7, anti-CD244 FITC, and anti-CD160 PE (Biolegend) before washing and flow cytometric analysis. Memory subset analysis utilized the same panel but included anti-CCR7 FITC (R&D systems) and anti-CD45RA AF700 (Biolegend) and omitted anti-CTLA4, anti-CD244, and anti-CD160. Example flow plots can be found in (Figure S1).

Parry H.M., Mirajkar N., Cutmore N., Zuo J., Long H., Kwok M., Oldrieve C., Hudson C., Stankovic T., Paneesha S., Kelly M., Begum J., McSkeane T., Pratt G, & Moss P. (2019). Long-Term Ibrutinib Therapy Reverses CD8+ T Cell Exhaustion in B Cell Chronic Lymphocytic Leukaemia. Frontiers in Immunology, 10, 2832.

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Cytokine and Degranulation Assay for T Cells

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Following identification of CMV positive and negative donors, 2 × 10⁶ cells were incubated at 37°C for 5 h with either PMA and ionomycin, CMV peptide mix (10 (link)) or EBV peptide mix at 10 μg/ml (Supplementary Table 6), along with protein cocktail inhibitor mix (eBiosciences). Live/dead red dye (Invitrogen), anti-CD3 APC-Cy7 (Biolegend) anti-CD8 Amcyan (BD Biosciences), were then applied before fixation and permeabilisation and IFN-γ AF700 (Biolegend) and TNF-α PE-Cy7 staining (eBioscience). Example flow plots can be found in supplementary (Figure S1). For assessing immune cell activation and cytotoxic degranulation, 2 × 10⁶ cells were stimulated with either the above peptide mixes overnight at 37°C or a cell stimulation cocktail (Invitrogen) for 5 h. At the time of stimulation, CD107a FITC (Biolegend), along with brefeldin A and monensin was incorporated into the stimulation panel (example staining of CD107a can be found in supplementary).

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Flow Cytometry Analysis of Adenovirus-Infected PBMCs

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After 24 hours, PBMCs infected with Ad5-GFP or Ad19a/64-GFP were washed twice with wash buffer (PBS with 1% FCS and 1 mg/ml NaN₃) and stained for 30 minutes with the following antibodies: anti-CD3-APC-Cy7 (Biolegend, clone SK7), anti-CD4-BV421 (Biolegend, clone RPA-T4), anti-CD8-AmCyan (BD Biosciences, clone SK1), anti-CD14-PE (Biolegend, clone 63D3), anti-CD19-APC (Biolegend, clone HIB19) and anti-CD56-PerCP (Biolegend, clone HCD56, all antibodies were diluted 1:60 in PBS). After staining, cells were washed twice with wash buffer and analyzed on a FACS Canto II device.

Kiener R., Fleischmann M., Schwegler C., Ruzsics Z., Thirion C., Schrödel S., Asbach B, & Wagner R. (2018). Vaccine vectors based on Adenovirus 19a/64 exhibit broad cellular tropism and potently restimulate HCMV-specific T cell responses ex vivo. Scientific Reports, 8, 1474.

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Phenotypic and Functional Profiling of T Cells

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For suppression assays, cells were washed with 0.1% (w/v) sodium azide/phosphate-buffered saline (Mediatech Cellgro) on day 7 of in vitro stimulation and were stained with anti-CD4 PECy5 (BD Pharmingen), anti-CD8 Pacific Blue (Biolegend), and anti-CD25 APCCy7 (BD Pharmingen. For surface phenotyping of cells, bulk PBMCs and enriched CD8+ T cells were stained with anti-CD3 Alexa 700 (BD Pharmingen), anti-CD8 AmCyan (BD Biosciences), anti-CD27 APCCy7 (Biolegend), anti-CD28 APC (BD Pharmingen), CD45RO Pacific Blue (Biolegend), anti-CD62L PECy5 (BD Pharmingen), and anti-CD57 PE (Southern Biotech). For intracellular staining of cytokines, cells were initially activated with 1 μL of leukocyte activation cocktail (BD Pharmingen) for 5 hours. Cells were surface stained with anti-CD8 APC (BD Biosciences), anti-CD4 PECy5 (BD Pharmingen) and anti-CD25 APCCy7 (BD Pharmingen) and permeabilized as described previously. Intracellular staining was performed using anti-IFNγ PECy7 (BD Pharmingen), anti-IL-17A PE (Ebioscience), anti-Granzyme B Alexa 700 (BD Pharmingen) and anti-Perforin Pacific Blue (BD Pharmingen). All cells were resuspended in 1% paraformaldehyde (Electron Microscopy Sciences, Hatfiled, PA) for FACS analysis. Flow cytometric data were acquired on a 4-Laser, 17-color LSRII using FACSDiva software (Becton Dickinson). CFSE was detected in the FITC channel on the LSR.

Cunnusamy K., Baughman E.J., Franco J., Ortega S.B., Sinha S., Chaudhary P., Greenberg B.M., Frohman E.M, & Karandikar N.J. (2014). Disease Exacerbation of Multiple Sclerosis is Characterized by Loss of Terminally Differentiated Autoregulatory CD8+ T cells. Clinical immunology (Orlando, Fla.), 152(0), 115-126.

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