Flow Cytometry: Rabbit IgG Isotype Control [PE] [NBP2-24983] - FLOW analysis of Mouse CD4+ T cells that were stimulated with anti-CD3/CD28 beads and insulin (1ug/mL) for 5 days in culture media with additional glucose ...read more
Flow Cytometry: Rabbit IgG Isotype Control [PE] [NBP2-24983] - PBMCs fixed and permeabilized using the Novus intracellular staining kit and stained with 1 ug of TLR7-PE conjugate (red) or rabbit IgG-PE conjugate ...read more
1:100 dilution in 1X Permeabilization buffer (Foxp3 Staining Buffer Set from eBiosciences), 30 minutes incubation at 21°C
LSR II Flow Cytometer (BD); Controls - Fluorescence Minus One/FMO and Glut1 stained Mouse CD4+ T Cells (NB110-39113PE, Orange)
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Cells fixed and permeabilized with Foxp3 Staining Buffer set (eBioSciences #00-5523-00) for intracellular staining
One wash with 1X Permeabilization buffer (eBiosciences) and one wash with FACS buffer (1x PBS, 1% BSA, 0.1% sodium azide)
Murine spleens were harvested and CD4+ T cells were isolated via negative selection. CD4+ T cells were stimulated with anti-CD3/CD28 beads and insulin (1μg/mL) for 5 days in culture media with additional glucose provided. Cells were washed and beads removed prior to staining. Cells were stained for surface antigens, washed, then fixed and permeabilized (via Foxp3 Staining Buffer set (eBioSciences #00-5523-00)) for intracellular staining (Novus informed me that the epitope is intracellular for this antibody). Cells were washed again before being ran on LSR II Flow Cytometer. Data was analyzed via FlowJo (Tree Star). Cells were gated, in order, on lymphocytes, single cells, live cells, and CD4+ cells. Test samples were compared to both isotype control, as well as FMO control.
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