Figure 2.
Depletion of circulating lymphocyte numbers after repeated bleeding is quantified by IVFC. T cells were isolated and purified from the lymph nodes and spleen, labeled with 10 μM of DiD, and injected intravenously into syngeneic BALB/c mice at a dose of 5 × 106 cells per mouse. After overnight equilibrium, in vivo flow cytometry was carried out to detect the number of donor T cells in the peripheral blood of the recipient mice. The experimental mice (n= 6) were then bled by tail snip and ~100 μl of blood was collected. Four hours later, the circulating fluorescently labeled donor T cells were enumerated again by in vivo flow cytometry. The procedure was repeated at 8 and 24 hours except that at these time points 50 μl of blood were collected, followed by in vivo flow cytometric analysis 4 hours later. Control mice (n=6) were not bled. The results are shown as the relative cell counts of circulating cells per minute. **P<0.01.