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).
Allophycocyanin apc labeled anti mouse cd4
Manufactured by Thermo Fisher Scientific
Allophycocyanin (APC)-labeled anti-mouse CD4 is a fluorescently-tagged antibody that binds to the CD4 surface protein on mouse T cells. It is used for the detection and analysis of CD4+ T cells in flow cytometry applications.
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Lab products found in correlation
Pe labeled anti mouse cd3, by Thermo Fisher Scientific (5 mentions)
System 2 software, by Beckman Coulter (4 mentions)
Fluorescein isothiocyanate fitc labeled anti mouse cd8, by Thermo Fisher Scientific (4 mentions)
Facscan flow cytometer, by BD (3 mentions)
Fitc labeled anti mouse cd8, by Thermo Fisher Scientific (1 mentions)
Facscan, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
6 protocols using allophycocyanin apc labeled anti mouse cd4
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
Lymphocyte Surface Marker Analysis
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Lymphocytes were isolated from spleen, as described above. According to the method described in our previous studies [3 (link), 40 (link)], surface staining was performed by incubation 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). The cell suspension was then fixed with FACScan buffer (PBS containing 1% BSA and 0.1% sodium azide) and 2% paraformaldehyde. All data were acquired through a FACScan flow cytometer (BD Biosciences, USA). The analysis was performed with the data from three independent experiments.
4
Phenotypic Analysis of Splenocytes
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For phenotypic analysis of splenocytes, a single cell suspension was prepared as described above, and 1 × 106 cells in 50 μL were delivered to each tube already containing 10 μL of Allophycocyanin (APC)-labeled anti-mouse CD4, 20 μL of phycoerythrin (PE)-labeled anti-mouse CD8, or 20 μL of fluorescein isothiocyanate (FITC)-labeled anti-mouse CD3 antibodies (all from eBioscience) and incubated at 4 °C for 20 min in the dark. After washing, the cells were fixed with FACScan buffer (PBS containing 1% BSA and 0.1% sodium azide) and 2% paraformaldehyde. The fluorescence profile of each sample (at least 10,000 cells) was analyzed on FACS-Calibur flow cytometer (BD Biosciences) using SYSTEM II software (Coulter).
5
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).
6
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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