For the simultaneous detection of cell surface expressed CD4, CD8 and CD3, a triple labelling technique was used. Thymocyte samples were incubated with monoclonal antibody cocktails for 30 min in 100 ml binding buffer on ice (PBS containing 0.1% NaN3 and 0.1% BSA), then washed twice in PBS, and finally resuspended in 500 ml 0.1% buffered PFA (paraformaldehyde) in PBS. For staining the following monoclonal antibodies were used: phycoerythrin (PE) conjugated rat anti-mouse CD4, CyChrome (CyC) conjugated rat anti-mouse CD8 and FITC conjugated rat anti-mouse CD3, all purchased from BD Pharmingen, CA. Samples were measured and analysed in a FACSCalibur flow-cytometer (Becton Dickinson, San Jose, CA), using the CellQuest software. Generally 10.000 events were recorded. Thymocytes were gated according to their size and granularity on forward and side scatter dot plots. The gate set on untreated control living thymocytes was used for the analysis of all samples. We used fluorescent dot plots for both comparing the different samples and for calculating the ratio of positively stained cells.
Phycoerythrin pe conjugated rat anti mouse cd4
Manufactured by BD
Phycoerythrin (PE) conjugated rat anti-mouse CD4 is a fluorescently labeled antibody that binds to the CD4 antigen expressed on the surface of mouse T helper cells.
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2 protocols using phycoerythrin pe conjugated rat anti mouse cd4
1
Triple Immunofluorescence Staining of Thymocytes
2
Adoptive Transfer of CD4+CD25- Cells
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Spleens harvested from female WT and Lcn2KO mice were crushed using frosted glass slides and washed with fluorescence-activated cell sorter (FACS) buffer (1× phosphate-buffered saline [PBS] with 2% FBS). After pelleting the cells, red blood cells (RBCs) were removed using RBC lysis buffer (Sigma) according to the manufacturer’s protocol. Subsequently, the cells were resuspended in 2 mL FACS buffer, filtered through a 70-μm nylon cell strainer, and incubated with Phycoerythrin (PE)-conjugated rat anti-mouse CD4 and BB515-conjugated rat anti-mouse CD25 (BD Biosciences, San Diego, CA). CD4+CD25- cells were sorted to more than 99.5% purity using a BD FACSAria III cell sorter. These cells were transferred (0.5 × 106 cells/mouse) to 4-week-old female recombination-activating gene 1 (Rag1)KO mice via tail vein injection and colitis was monitored after 8 weeks of CD4+CD25- cell administration.
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