After viability determination of harvested lymphocytes from each group using 0.04% trypan blue (viability > 90%) and adjusted cell concentration to 1 × 106 cells/ml in PBS containing 2% FBS, the cells were incubated with surface markers including phycoerythrin (PE)-labeled anti-mouse CD3, Allophycocyanin (APC)-labeled anti-mouse CD4 and fluorescein isothiocyanate (FITC)-labeled anti-mouse CD8 (eBioscience) at 4°C for 30 min in the dark. Followed by washed by 2 ml PBS and then fixed with FACScan buffer (PBS containing 1% FCS and 0.1% Sodium azide) and 2% paraformaldehyde, the cultures were analyzed of fluorescence profiles on a FACScan flow cytometer (BD Bio-sciences) by SYSTEM II software (Coulter).
Fluorescein isothiocyanate fitc labeled anti mouse cd8
Manufactured by Thermo Fisher Scientific
Fluorescein isothiocyanate (FITC)-labeled anti-mouse CD8 is a monoclonal antibody conjugated with the fluorescent dye FITC. It is designed for the detection and analysis of CD8-positive cells in mouse samples using flow cytometry or fluorescence microscopy.
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4 protocols using fluorescein isothiocyanate fitc labeled anti mouse cd8
1
Flow Cytometry Analysis of Immune Cells
2
Quantifying T cell subsets by flow cytometry
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The concentration of the purified splenocytes were adjusted into 1× 105 cells/mL. Then the phycoerythrin (PE)-labeled anti-mouse CD3 (eBioscience) (5 μg/mL), Allophycocyanin (APC)-labeled anti-mouse CD4 (eBioscience) (5 μg/mL) and fluorescein isothiocyanate (FITC)-labeled anti-mouse CD8 (eBioscience) (5 μg/mL) antibodies were used to stain the T cell subclasses (CD4+ and CD8+) for 30 min at 4 °C. After washing by PBS, the cultures were fixed with FACScan buffer (1% FCS and 0.1% Sodium azide in PBS) and 2% paraformaldehyde. The T cell subclasses were measured for fluorescence profiles on a FACScan flow cytometer (BD Bio-sciences) and analyzed by SYSTEM II software (Coulter).
3
Flow Cytometric Analysis of T Cells
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Analyses of CD4+ and CD8+ T lymphocytes were performed according to our previous study [25 (link)]. The percentages of CD4+ and CD8+ T lymphocytes were determined using flow cytometry with staining by the surface markers including phycoerythrin- (PE-) labeled anti-mouse CD3 (eBioscience), allophycocyanin- (APC-) labeled anti-mouse CD4 (eBioscience), and fluorescein isothiocyanate- (FITC-) labeled anti-mouse CD8 (eBioscience) antibodies. All the samples were analyzed regarding fluorescence profiles on a FACScan flow cytometer (BD Biosciences) by SYSTEM II software (Coulter).
4
Multicolor Flow Cytometry Immunophenotyping
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The splenic lymphocytes were collected as described above [18] ,
The viability was determined with 0.04% trypan blue (viability > 90%) and alive cell concentration was adjusted to 1 × 10 6 cells/ml in PBS containing 2% FBS, and the lymphocytes were incubated with 3 different surface markers at 4 °C for 30 min in the dark [19, 20] , including phycoerythrin (PE)-labeled anti-mouse CD3, allophycocyanin (APC)-labeled antimouse CD4 and fluorescein isothiocyanate (FITC)labeled anti-mouse CD8 (eBioscience). Then the cells were fixed with FACScan buffer (PBS containing 1% FCS and 0.1% Sodium azide) and 2% paraformaldehyde. The specific cells were analyzed according to the surface markers (CD3, CD4 and CD8) through a FACScan flow cytometer (BD Biosciences, USA).
The viability was determined with 0.04% trypan blue (viability > 90%) and alive cell concentration was adjusted to 1 × 10 6 cells/ml in PBS containing 2% FBS, and the lymphocytes were incubated with 3 different surface markers at 4 °C for 30 min in the dark [19, 20] , including phycoerythrin (PE)-labeled anti-mouse CD3, allophycocyanin (APC)-labeled antimouse CD4 and fluorescein isothiocyanate (FITC)labeled anti-mouse CD8 (eBioscience). Then the cells were fixed with FACScan buffer (PBS containing 1% FCS and 0.1% Sodium azide) and 2% paraformaldehyde. The specific cells were analyzed according to the surface markers (CD3, CD4 and CD8) through a FACScan flow cytometer (BD Biosciences, USA).
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