Benoist1 project protocol

Survey of regulatory T cells (Treg) of the thymus and spleen in males of 33 inbred strains of mice   (2007)

Benoist C, Mathis D, Satpathy A, Feuerer M
With: Jiang W, Holler P, Campbell C




Benoist1_Protocol

Project protocol — Contents
Workflow and sampling
Equipment
Reagents, supplies, and solutions
Procedure
Data
References



Workflow and sampling

Workflow

Test
Procedure performed
Age
Data collected
1
 Harvest thymus and spleen
5-6 wks
flow cytometry

Equipment

  • Dissecting instruments
  • FACS -- Coulter Analyzer (fluorescence-activated cell sorting)

Reagents, supplies, solutions

  • 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

Procedure

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+.