Figure 2.

Intracellular expression of Ki-67 and Granzyme B in CD3⁻CD5⁻CD21⁻, CD3⁺CD5^dimCD21⁻, and CD3⁺CD5^brightCD21⁻ cell compartments, and analysis of cell death frequency during PBMC culture for 21 days. (A) Flow cytometric analysis of Ki-67 expression (n = 9). (B) Changes in the frequency of each cell population within Ki-67-expressing cells during culture for 21 days (n = 9). (C) Changes in the frequency of Granzyme B intracellular expression in CD3⁻CD5⁻CD21⁻, CD3⁺CD5^dimCD21⁻, and CD3⁺CD5^brightCD21⁻ cell populations during PBMC culture for 21 days. Expression level of Granzyme B represents relative mean fluorescence intensity (rMFI) (n = 9). (D) Representative flow cytometry data of the frequency of Granzyme B intracellular expression in each cell population during culture for 21 days (n = 9). (E) The frequency of cell death during culture for 21 days. Whole cells were stained with annexin V/propidium iodide (PI) on days 7, 10, 14, and 21 of culture. The population of cells that were negative for both annexin V and PI was defined as living cells. Annexin V⁺/PI⁻ cells were classified as early apoptotic cells, and double-positive cells were classified as late apoptotic cells. Annexin V⁻/PI⁺ cells were classified as dead cells (n = 5). (F) Representative flow cytometry data of annexin V/PI staining of cells during culture to evaluate the frequency of cell death frequency among the total cell population (n = 5).