For T cell isolation, blood samples were diluted with 1 volume of PBS 2% FBS, then the diluted blood was added dropwise to 1 volume of Lymphoprep (Stemcell Technologies Inc, Cat# 07811) at room temperature. Samples were centrifuged at 800g for 20 minutes at 22 °C, then the PBMCs layer was harvested, washed two times in PBS 2% FBS, and resuspended in PBS 2% FBS. CD8+ T cells were then purified by two subsequent rounds of isolation: first, T cells were enriched using the Pan T Cell Isolation kit (Miltenyi Biotec, Cat# 130-096-535); then, negative CD8 T cell isolation was performed with the CD8+ T Cell Isolation Kit, human (Miltenyi Biotec, Cat# 130-096-495) or negative CD4 T cell isolation was performed with the CD4+ T Cell Isolation Kit, human (Miltenyi Biotec, Cat# 130-096-533). Both enrichment steps were performed using an AutoMACS machine. For both mouse and human cells, CD8 T cell purity was assessed by FACS staining using mouse or human anti-CD3 and anti-CD8 antibodies at 1:100 dilution (PE/Cyanine7 anti-human CD3, BioLegend, # Cat317333; APC/Cyanine7 anti-mouse CD8a, BioLegend, Cat# 100714; FITC anti-human CD3, ThermoFisher Scientific, Cat#11-0038-42; PE/Cy7 anti-human CD8, Biolegend, Cat# 344712). Human CD4+ T cell purity was assessed using the A700 anti-human CD4 antibody, Biolegend, Cat# 317426 at 1:100 dilution.
Apc cyanine7 anti mouse cd8a
Manufactured by BioLegend
APC/Cyanine7 anti-mouse CD8a is a fluorescent-conjugated antibody that binds to the CD8a surface antigen expressed on mouse T cells. It can be used for the identification and analysis of CD8+ T cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti human cd3 fitc, by Thermo Fisher Scientific (2 mentions)
Cd8 t cell isolation kit, by Miltenyi Biotec (2 mentions)
Cd4 t cell isolation kit, by Miltenyi Biotec (2 mentions)
Pan t cell isolation kit, by Miltenyi Biotec (2 mentions)
Lymphoprep, by STEMCELL (2 mentions)
Pe cy7 anti human cd8, by BioLegend (2 mentions)
A700 anti human cd4 antibody, by BioLegend (2 mentions)
Pe cyanine7 anti human cd3, by BioLegend (2 mentions)
Fitc anti mouse cd69, by BioLegend (1 mentions)
Cytoflex s flow cytometer, by Beckman Coulter (1 mentions)
Fitc anti mouse cd45, by BioLegend (1 mentions)
7 aad viability staining solution, by BioLegend (1 mentions)
4 protocols using apc cyanine7 anti mouse cd8a
1
Isolation of CD4+ and CD8+ T Cells
2
Comprehensive T Cell Characterization
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Biomaterials Science stained by APC/Cyanine 7 anti-mouse CD8a (100714, Biolegend) and FITC anti-mouse CD69 (104505, Biolegend) for flow cytometry assay. For the proliferation assay, the cells were stained with CFSE at a final concentration of 2 μM at 37 °C for 15 min. Then, the cells were washed twice and resuspended in 1640 medium containing IL-2, followed by the addition of anti-CD3/CD28 beads at a bead-to-cell ratio of 1 : 1. After 48 h, the cells were collected and analysed by flow cytometry. For the cytokine secretion of OT-1 T cells, the cells were resuspended using 1640 complete medium containing 50 ng mL -1 PMA, 5 μg mL -1 BFA, and 1.5 μg mL -1 ionomycin. The cells were seeded into suspension 6-well cell culture plates at a density of 1 × 10 6 cells per mL, and cultured at 37 °C under a humidified atmosphere of 5% CO 2 for 4 h. The cells were then stained with APC/Cyanine 7 anti-mouse CD8a followed by PE anti-mouse TNF-α, APC anti-mouse IFN-γ, and FITC antimouse GzmB for flow cytometry assay. The cytotoxic efficiency of OT-1 T cells was measured by quantifying the release of endogenous lactate dehydrogenase (LDH) from B16-Ova cells using the LDH cytotoxicity assay kit according to the manufacturer's instructions. The effective target cell (E/T) ratio was 10 : 1.
3
Flow Cytometric Analysis of Murine Leukocytes
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Mice were deeply anesthetized using ketamine and xylazine. Then, the mouse’s chest was opened, the heart was exposed, and a needle was inserted into the right ventricle to collect blood. Blood was collected and treated with red blood cell lysis buffer to collect leukocytes. Leukocytes from the blood and BALF were incubated with fluorescent antibodies (5μL/test) for 30 min on ice in a flow buffer protected from light. Subsequently, the samples were washed with phosphate-buffered saline (PBS) twice and centrifuged to remove the supernatant. The samples were resuspended in 200 μL flow buffer and then ran on a 13-laser CytoFLEX S Flow Cytometer (Beckman). Flow cytometry standard (FCS) files were analyzed using Flowjo v10.6.2 software (BD). The flow cytometry antibodies as follow: FITC anti-mouse CD45 (Biolegend, Cat #103108), Brilliant Violet 510™ anti-mouse CD3 (Biolegend, Cat #100234), APC/Cyanine7 anti-mouse CD8a (Biolegend, Cat #100714), Alexa Fluor® 700 anti-mouse CD4 (Biolegend, Cat #100430), Brilliant Violet 605™ anti-mouse/human CD11b (Biolegend, Cat #101257), Brilliant Violet 650™ anti-mouse Ly-6G/Ly-6C (Gr-1)(Biolegend, Cat #108442),7-AAD Viability Staining Solution (Biolegend, Cat #420403).
4
Isolation of CD4+ and CD8+ T Cells
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