Encyclopedia of Experiments: Immunology
A subscription to JoVE is required to view this content.
Transcript
For immunization of the adoptively transferred animals, shave the fur over the gluteus superficialis of the OT-I T cell transplanted mouse, and use a 25-gauge needle to subcutaneously inject 50 microliters of endotoxin-free ova into the shaved area.
It's important to use endotoxin-free ova because traces of endotoxin in ova can lead you to false results.
48 hours after the immunization, harvest the inguinal lymph nodes and spleens from each recipient animal according to standard protocols. and generate individual single-cell suspensions for each lymph node and spleen sample, as just demonstrated. Collect the cells by centrifugation, and resuspend the pellets in 100 microliters of PBS per sample.
To stain the samples for flow cytometric analysis, incubate the cells with 100 microliters of fluorophore-conjugated primary antibody master mix cocktail for 30 minutes at 4 degrees Celsius. At the end of the incubation, wash the cells two times in 10 milliliters of PBS, and resuspend the samples in 0.5 to 1 ml of PBS per tube.
Filter the cells through 70 to 100-micrometer strainers into 5-millimeter flow cytometry analysis tubes, and acquire the single standing compensation controls, as well as the samples in the flow cytometer. To gate the samples, open a forward-scatter height versus forward-scatter area plot and a side-scatter wide versus side-scatter area plot and gate to exclude the doublets.
Generate a FITC versus autofluorescence plot in order to discriminate the population of true CSFE stain cells from the high autofluorescence cells. Gate these OT1-CSFE stain cells as the populations that extend mostly on the FITC axis.
To distinguish cells derived from donor versus recipient animals, gate the Thy1.1 plus CD90.1 plus CD8+ T cells, and regate this CD8+ population to exclude the CD4+ T cells. Then, display this proliferated CD8 T cell population in a histogram, according to their CSFE expression, and gate the proliferated populations from a 10 to the second intensity up to the undivided control population intensity. Acquire at least 5,000 events for the compensation controls and 10,000 events for each sample at a low or medium flow analysis rate.
