American Society of Hematology

Fig. 1.

$Fig. 1. Fractionation of T cells after apoptotic induction. / Activated T-cell populations exposed for 2 hours to CD2 or CD95 apoptotic induction and fractionated according to density on discontinuous Percoll gradients give rise to fractions progressively enriched in cells committed to apoptosis. (A) Homogeneous populations of large activated T cells (F2-I) obtained from the low buoyant density fractions of a first primary Percoll gradient were exposed for 2 hours to CD2 mAb (D66 +T111, 2 μg/mL) and were then fractionated again on secondary Percoll gradients to yield fractions F2-II, F3, F4, and F5 with decreasing cell volumes. Columns are mean cell volumes ± SD of determinations performed in 3 different experiments, as measured by using a Coulter counter and a channelyser. ⋄---⋄ indicates percent dead cells in the fractionated cell populations 2 hours after apoptotic induction, just after their recovery from the gradients, as assessed by trypan blue uptake and cell morphology (n = 40); ○---○, percent dead cells in fractionated cell populations replaced after washing in culture for 18 hours in IL-2 containing medium (n = 10); •---•, percent dead cells in cells exposed to 50 μmol/L BOC-D.fmk during apoptotic induction and during subsequent culture in IL-2 containing medium (n = 3); ▴---▴, percent dead cells similarly exposed to 50 μmol/L Z-VAD.fmk. Values are means ± SD. (B) Activated (F2-I) T cells exposed for various periods of time to anti-CD2 mAb. At each time point, the percentages of cells recovered in the F2-II (□---□), F3 (•---•), F4 (▵---▵), and F5 (○---○) Percoll fractions were estimated. It is seen that the percentages of F2-II cells progressively decreased within 2 hours, whereas the percentages of F3, and to a lesser extent, those of F4 and F5 cells concomitantly increased. After 3 hours, the percentages of F2-II cells were stabilized, but a further rise in F5 cells occurred at the expense of F3 and F4 cells. (C) Fractionation of F2-I cells treated for 2 hours with an anti-CD95 at 250 ng/mL (n = 10). Symbols are the same as in panel A. Cells treated in parallel with 50 μmol/L BOC-D.fmk did not shrink and were therefore recovered in majority in the low buoyant fraction (F2-II) of the secondary Percoll gradients (•). (D) Forward scatter (FSC) analysis showing the reduction of cell size from F2-II to F5. Numbers refer to the mean forward scatter. SSC, side scatter.$

Fractionation of T cells after apoptotic induction.

Activated T-cell populations exposed for 2 hours to CD2 or CD95 apoptotic induction and fractionated according to density on discontinuous Percoll gradients give rise to fractions progressively enriched in cells committed to apoptosis. (A) Homogeneous populations of large activated T cells (F2-I) obtained from the low buoyant density fractions of a first primary Percoll gradient were exposed for 2 hours to CD2 mAb (D66 +T11₁, 2 μg/mL) and were then fractionated again on secondary Percoll gradients to yield fractions F2-II, F3, F4, and F5 with decreasing cell volumes. Columns are mean cell volumes ± SD of determinations performed in 3 different experiments, as measured by using a Coulter counter and a channelyser. ⋄---⋄ indicates percent dead cells in the fractionated cell populations 2 hours after apoptotic induction, just after their recovery from the gradients, as assessed by trypan blue uptake and cell morphology (n = 40); ○---○, percent dead cells in fractionated cell populations replaced after washing in culture for 18 hours in IL-2 containing medium (n = 10); •---•, percent dead cells in cells exposed to 50 μmol/L BOC-D.fmk during apoptotic induction and during subsequent culture in IL-2 containing medium (n = 3); ▴---▴, percent dead cells similarly exposed to 50 μmol/L Z-VAD.fmk. Values are means ± SD. (B) Activated (F2-I) T cells exposed for various periods of time to anti-CD2 mAb. At each time point, the percentages of cells recovered in the F2-II (□---□), F3 (•---•), F4 (▵---▵), and F5 (○---○) Percoll fractions were estimated. It is seen that the percentages of F2-II cells progressively decreased within 2 hours, whereas the percentages of F3, and to a lesser extent, those of F4 and F5 cells concomitantly increased. After 3 hours, the percentages of F2-II cells were stabilized, but a further rise in F5 cells occurred at the expense of F3 and F4 cells. (C) Fractionation of F2-I cells treated for 2 hours with an anti-CD95 at 250 ng/mL (n = 10). Symbols are the same as in panel A. Cells treated in parallel with 50 μmol/L BOC-D.fmk did not shrink and were therefore recovered in majority in the low buoyant fraction (F2-II) of the secondary Percoll gradients (•). (D) Forward scatter (FSC) analysis showing the reduction of cell size from F2-II to F5. Numbers refer to the mean forward scatter. SSC, side scatter.

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