In order to assess receptors expression on activated CD4+ and CD8+ T cells, PBMC isolated from 4 patients and 4 controls were stimulated for 24 hours with anti-CD3/CD28 antibodies coated microbeads-Dynabeads Human T-Activator (Dynal, Oslo, Norway), according to the manufacturer's recommendations. Cells were cultured in RPMI-1640-GlutaMAX-I, supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin, and 100 microg/mL streptomycin (all purchased from Life Technologies, Carlsbad, CA). In order to identify CD4+ and CD8+ T lymphocytes, we incubated cells with a mixture of the following antibodies: PerCp-conjugated anti-CD3, APC-H7-conjugated anti-CD4, and APC-conjugated anti-CD8 antibodies. Activated cells were detected by incubating cells with FITC-conjugated anti-CD25 antibodies; all reagents were purchased from BD Biosciences. Cells were previously stained with anti-ETA and anti-ETB primary and secondary antibodies as previously described and samples were acquired on a FACSCanto cytometer FlowJo 8.8.2 software was used to analyse data. The variation in receptors surface exposure was expressed as the difference between activated cells MFI and unstimulated cells MFI (ΔΔMFI).
Facs canto cytometer
Manufactured by FlowJo
The FACS Canto cytometer is a flow cytometry instrument designed for cell analysis. It provides the core function of detecting and analyzing cells in a fluid sample as they pass through a laser beam. The cytometer measures various parameters of individual cells, such as size, granularity, and fluorescence, enabling researchers to identify and characterize different cell populations.
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Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 glutamax 1, by Thermo Fisher Scientific (1 mentions)
Cd11c pe cy7, by BD (1 mentions)
Fc blocking antibody, by BD (1 mentions)
Mouse serum, by Merck Group (1 mentions)
Ghost dye red 780, by Cytek Biosciences (1 mentions)
Pe anti human cd279 pd 1 eh12.2h7, by BioLegend (1 mentions)
3 protocols using facs canto cytometer
1
Assessing T Cell Receptor Expression
2
Uptake of OVA peptide by mouse dendritic cells
3
PD-1 Expression Quantification in Cells
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The protein levels were measured after different time points of incubation by ow cytometry in a FACS Canto cytometer and analyzed with FlowJo software (FlowJo, LLC). Cells were rinsed twice with PBS (1X) and resuspended with PBS and Ghost Dye Red 780 (1:1000 dilution, Tonbo Biosciences) for 30 min on ice. After that, cells were rinsed once with PBS-0.2% Bovine Serum Albumin (BSA) (PanReac AppliChem) and resuspended in 50 µl of this buffer with the PE anti-human CD279 (PD1) (EH12.2H7, 1:100 dilution, BioLegend) antibody for 30 min on ice. The analysis strategy was to compare the mean uorescence intensity between treated and untreated live cells for oxLDL experiments. For anti-CD69 monoclonal antibody engagement experiments, samples were incubated with mouse serum (1:100 dilution, Sigma-Aldrich) for 15 min before adding the same volume with ow cytometry antibodies. Percentage of positive cells for PD-1 were represented at different times.
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