Project protocol — Contents
Workflow and sampling
Reagents, supplies, and solutions
Test Procedure performed Age Data collected 1 Harvest thymus and spleen 5-6 wks flow cytometry
- Dissecting instruments
- FACS -- Coulter Analyzer (fluorescence-activated cell sorting)
- Monoclonal antibodies: BD-Pharmingen
- Gel Foam surgical sponges; Amersham Pharmacia, Piscataway, NJ; Supor 450 membrane, 0.45-µm pore size, Pall Gelman Laboratory, East Hills, NY)
- RPMI medium 1640 (10% FCS/50 µM 2-mercaptoethanol/2 mM L-glutamine/10 mM Hepes/nonessential amino acids/sodium pyruvate/penicillin/streptomycin)
- BDC-specific peptide mimotope at indicated concentrations (BDCmi, peptide 1040–63; ref. 44)
- 1.35 mM 2-deoxyguanosine
a. Organs are harvested and teased between glass microscope slides in RPMI to yield spleen and thymus cells.
b. Red cells are lysed and cell suspensions kept on ice.
c. Cells are counted, stained with monoclonal antibodies, and washed prior to FACS analysis.
Data collected by investigator
Percent of thymic CD4 T cells that are CD25hi and Foxp3+ and percent of spleenic CD4 T cells that are CD25hi and Foxp3+.