Chen1 project protocol

Invariant natural killer T-cells in peripheral blood, spleen, and thymus of 38 inbred strains of mice   (2011)

Chen Y, Serreze DV
With: Tsaih SW

Chen1 Protocol

Project protocol - Contents

Workflow and sampling

Data collected
1 Mice euthanized and tissues harvested (peripheral blood, spleen, thymus) -
2 Cell suspensions processed and analyzed Immune cell populations

Equipment and supplies

  • FACSCalibur flow cytometer (Becton Dickinson, San Jose CA)
  • CellQuest software

Reagents and solutions

  • alpha-galactosylceramide analog PBS57-loaded CD1d tetramers (NIH tetramer core facility)
  • anti-CD16/CD32 (clone 2.4G2) (BD Bioscience, San Diego CA)
  • anti-CD4 (clone RM4-5) (BD Bioscience, San Diego CA)
  • anti-TCR beta (clone H57-597) (BD Bioscience, San Diego CA)
  • propidium iodide

Procedure: Flow cytometry

    1. Red blood cell-depleted single cell suspensions are prepared from peripheral blood, spleen, and thymus.
    2. Cell suspensions are Fc-blocked at room temperature with anti-CD16/CD32 for 10 min and washed.
    3. Cell suspensions are incubated with an antibody cocktail containing CD1d/alpha-galactosylceramide analog-loaded tetramers, anti-CD4 and anti-TCR beta for 30 min at 4°C.
    4. Stained cells are washed and analyzed by flow cytometry (dead cells are excluded by propidium iodide).

Data collected by investigator

  • Percentage of TCR beta cells that are invariant NK T (iNKT) cells for peripheral blood, spleen, and thymus
  • Percentage of iNKT cells that express CD4 for peripheral blood, spleen, and thymus

Ratios were computed by MPD.