Cd8a apc efluor 780

Manufactured by Thermo Fisher Scientific

The CD8a-APC-eFluor 780 is a fluorescently-labeled antibody that binds to the CD8a surface marker. It is designed for use in flow cytometry applications to identify and quantify CD8a-positive cells in a sample.

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Lab products found in correlation

Cd4 apc efluor 780, by Thermo Fisher Scientific (2 mentions) Cd4 pe cf594, by BD (2 mentions) Cd19 bv711, by BD (2 mentions) Cd3 alexa fluor 647, by BioLegend (2 mentions) Facsverse flow cytometer, by BD (1 mentions) Cd4 percp cy5, by Thermo Fisher Scientific (1 mentions) Cd3 bv510, by BD (1 mentions) Cd19 fitc, by BioLegend (1 mentions) Sy3200 cell sorter, by Sony (1 mentions) Ack lysing buffer, by Thermo Fisher Scientific (1 mentions) Fixable viability stain 700, by BD (1 mentions) Mouse memory b cell isolation kit, by Miltenyi Biotec (1 mentions) Igg1 bv421, by BD (1 mentions) Cd4 efluor450, by Thermo Fisher Scientific (1 mentions) Fcs express 4, by De Novo Software (1 mentions) Foxp3 pe, by Thermo Fisher Scientific (1 mentions) Ifn γ pe, by BioLegend (1 mentions) Cd62l fitc, by BioLegend (1 mentions) Lsrfortessa, by BD (1 mentions) Cd45 efluor450, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

EFluor 780 (389 citations) APC-eFluor 780 (35 citations) CD8a-APC (14 citations) Anti-CD8a-APC-eFluor 780 (clone RPA-T8; (3 citations)

7 protocols using cd8a apc efluor 780

Nickel-Specific T Cell Proliferation

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Nickel‐specific T cell proliferation was assessed by flow cytometry. Acquisition and analysis were performed on a BD FACSVerse flow cytometer and associated FACSuite software using the gating strategy depicted in Figure 1. Ten thousand events were collected. Lymphocytes were gated for CD3⁺, CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells. Cell surface marker staining was performed using the following three monoclonal antibodies: CD3 BV510 (BD), CD4 PerCP‐Cy5.5 (Thermo Fisher) and CD8a APC‐eFluor 780 (Thermo Fisher). The results were expressed as the stimulation index (SI), which is calculated as the ratio of the percentage of CFSE^low/neg CD3⁺, CD3⁺CD4⁺ or CD3⁺CD8⁺ T‐lymphocytes upon nickel stimulation to the percentage of CFSE^low/neg CD3⁺, CD3⁺CD4⁺ or CD3⁺CD8⁺ T lymphocytes without antigen.

de Graaf N.P., Bontkes H.J., Roffel S., Kleverlaan C.J., Rustemeyer T., Gibbs S, & Feilzer A.J. (2020). Non–heat inactivated autologous serum increases accuracy of in vitro CFSE lymphocyte proliferation test (LPT) for nickel. Clinical and Experimental Allergy, 50(6), 722-732.

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Flow Cytometric Analysis of Antigen-Specific B-cells

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B-cell analysis using flow cytometry was carried out as described (54 ). Briefly, single-cell suspensions were prepared from mouse spleens using mechanical dissociation, and red blood cells were removed using ACK lysing buffer (Gibco). The white blood cell preparation was enriched for IgG+ B-cells using the negative selection protocol in a mouse memory B-cell isolation kit (Miltenyi). The following commercial reagents were used to stain enriched splenocytes: CD4-APC-eFluor 780 (clone: RM4–5), F4/80-APC-eFluor 780 (clone: BM8), CD8a-APC-eFluor 780 (clone: 53–6.7), Ly-6G-APC-eFluor 780 (clone: RB6–8C5), IgM- APC-eFluor 780 (clone: II/41) (Thermo Fisher Scientific), CD19-FITC (clone: 6D5) (Biolegend), IgG1 BV421 (clone: X40) and IgG2 BV421 (clone: R19–15) (BD Bioscience). SARS-2 RBD-APC and Rs4081 RBD-PE for used to identify antigen-specific B-cells. Cell viability was analyzed with Fixable Viability Stain 700 (BD Bioscience). Stained cells were analyzed with a SY3200 Cell Sorter (Sony) configured to detect 6 fluorochromes. 2,000,000 events were collected per sample and analyzed via FlowJo software (TreeStar).

Cohen A.A., Gnanapragasam P.N., Lee Y.E., Hoffman P.R., Ou S., Kakutani L.M., Keeffe J.R., Wu H.J., Howarth M., West A.P., Barnes C.O., Nussenzweig M.C, & Bjorkman P.J. (2021). Mosaic nanoparticles elicit cross-reactive immune responses to zoonotic coronaviruses in mice. bioRxiv.

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Flow Cytometry Analysis of Lymphocytes

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Flow cytometry was performed using a BD Biosciences LSRFortessa. For direct ex vivo staining of lymphocytes, 1×10⁶ to 2×10⁶ cells were resuspended in 100 mL of FACS buffer (PBS þ 1% Fetalclone III), and Fc receptors were blocked for 10 to 15 minutes at 4 C with 1 µg/sample of 2.4G2 mAb (CD16 and CD32 blockade) and 1 µg/sample of mouse IgG (ThermoPierce). Cells were fluorescently labeled with antibodies for 30 minutes at 4 C, washed, and resuspended in fixation buffer (2% formaldehyde in PBS), or intracellularly stained according to the manufacturer's protocol (eBiosciences). The following fluorescently labeled anti- mouse mAbs were used for flow cytometry: IFNγ -FITC, IFNγ-PE, CD44-PerCP-Cy5.5, CD4-APC, CD8a-APC (BioLegend); CD62L- FITC, FoxP3-PE, TNFα-PerCP-eFluor 710, CD45-eFluor 450, CD4-eFluor450, CD62L-eFluor 450, CD4-APC-eFluor 780, and CD8a-APC-eFluor 780 (eBioscience); active caspase-3-PE (BD Biosciences). Flow cytometry data were analyzed with FCS Express 4 (De Novo Software).

Higuchi T., Flies D.B., Marjon N.A., Mantia-Smaldone G., Ronner L., Gimotty P.A, & Adams S.F. (2015). CTLA-4 Blockade Synergizes Therapeutically with PARP Inhibition in BRCA1-Deficient Ovarian Cancer. Cancer immunology research, 3(11), 1257-1268.

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Immune Cell Profiling by Flow Cytometry

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Blood cells from whole blood were incubated with fluorochrome-conjugated antibodies for 30 min at room temperature. After incubation, samples were washed with PBS containing 5% FBS, after which Optilyse C (Beckman Coulter, A11895) was added to lyse erythrocytes. Antibodies against the following proteins were used: CD3e-PE (Clone 145-2C11; 12-0031-82), CD8a-APC-eFluor780 (Clone: 53-6.7; 47-0081-82), CD11b-AlexaFluor488 (Clone: M1/70; 53-0112-82), CD19-eFluor 450 (Clone: 1D3; 48-0193-82) and F4/80-PE-Cy7 (Clone: BM8; 25-4801-82) purchased from eBioscience. From BD Bioscience CD4-PerCP (Clone RM4-5; 553,052), and Ly6G-APC (Clone 1A8; 560599) were used. The Gallios Flow Cytometer (Beckman Coulter) was used to measure cell fluorescence and all analyses were performed with Kaluza Analysis Software (Beckman Coulter, version 1.3). Results are expressed as the number of immune cells per milliliter. To determine this concentration, the number of cells determined in a fixed volume of blood (20 μL at baseline and 50 μL at termination) was multiplied by 50 and 20, respectively, to estimate the number of cells in 1 mL. The gating strategy can be found in the Supplementary Figure S2.

Kilgallen A.B., van den Akker F., Feyen D.A., Crnko S., Snijders Blok C.J., Gremmels H., du Pré B.C., Reijers R., Doevendans P.A., de Jager S.C., Sluijter J.P., Sampaio-Pinto V, & van Laake L.W. (2022). Circadian Dependence of the Acute Immune Response to Myocardial Infarction. Frontiers in Pharmacology, 13, 869512.

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Isolation and Characterization of CD20+ and CD20- Cells

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Buffy coat samples were washed with PBS 2% FCS and stained for 60 min at 4 °C with Rituximab CD20-FITC, CD3-APC (eBioscience), CD8a -APC-eFluor780 (eBioscience), CD45RO- PE-Cy7 (eBioscience), CCR7-BV421 (BD), and markers CD4-PE (eBioscience), CD56-PE (eBioscience) and CD19-PE (BD). Cells were washed and filtered using a 35 µm strainer (Falcon). Propidium Iodide (1 µg/ml) was used for exclusion of dead cells. From the CD3 + CD8 + CD45RO + CCR7- population, 1000 live CD20 + and 1000 live CD20-cells were sorted using a Beckman Coulter MoFlo Astrios cytometer. UltraComp eBeads (Thermo Fisher Scientific) were used as compensation controls.

Vlaming M., Bilemjian V., Freile J.Á., Lourens H.J., van Rooij N., Huls G., van Meerten T., de Bruyn M, & Bremer E. (2021). CD20 positive CD8 T cells are a unique and transcriptionally-distinct subset of T cells with distinct transmigration properties. Scientific Reports, 11, 20499.

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Isolation and Flow Cytometry of PBMCs

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The isolation of peripheral blood mononuclear cells (PBMCs) and flow-based transmigration assays were performed in a 3D BioFlux flow chamber device (Fluxion Bioscience, San Diego, CA) as previously described.^{30 (link)} The migrated cells were enumerated by a hemocytometer and then normalized to migrated cell numbers determined by flow cytometry. After collection, cells were fixed for 10 minutes in 1% paraformaldehyde at room temperature and washed in phosphate buffered saline + 0.1 mM ethylenediaminetetraacetic acid, followed by blocking in mouse IgG. Cells were labeled with anti-human CD45 eFluor450, CD8a APC-eFluor780 (eBioscience, San Diego, CA), CD3Alexa Fluor 647 (BioLegend, San Diego, CA), CD19 BV711, and CD4 PE-CF594 (BD Biosciences). Data were acquired using BD FACSCanto II (BD Biosciences) and analyzed by FlowJo software (v.10.4.1; Treestar, Ashland, OR).

Takeshita Y., Fujikawa S., Serizawa K., Fujisawa M., Matsuo K., Nemoto J., Shimizu F., Sano Y., Tomizawa-Shinohara H., Miyake S., Ransohoff R.M, & Kanda T. (2021). New BBB Model Reveals That IL-6 Blockade Suppressed the BBB Disorder, Preventing Onset of NMOSD. Neurology® Neuroimmunology & Neuroinflammation, 8(6), e1076.

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Multiparameter Flow Cytometry Immunophenotyping

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Input and migrated cells were analyzed by flow cytometry. After collection, cells were fixed for 10 minutes in 1% paraformaldehyde at room temperature, washed in PBS/0.1 mM EDTA, followed by blocking in mouse IgG. Cells were labeled with anti-human CD45 efluor450, CD8a APC-efluor780 (eBiosciences, San Diego, CA), CD3 Alexa Fluor 647, CD14 BV605 (BioLegend, San Diego, CA), CD19 BV711, CD4 PE-CF594 (BD Biosciences), and CD16 PE (R&D Systems). Data were acquired on a BD LSRFortessa SORP flow cytometer running Diva6, and analyzed in FlowJo 9.7.5 (Tree Star, Ashland, OR).

Takeshita Y., Obermeier B., Cotleur A.C., Spampinato S.F., Shimizu F., Yamamoto E., Sano Y., Kryzer T.J., Lennon V.A., Kanda T, & Ransohoff R.M. (2016). Effects of neuromyelitis optica–IgG at the blood–brain barrier in vitro. Neurology® Neuroimmunology & Neuroinflammation, 4(1), e311.

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