Repair of perforin pores at the plasma membrane restricts granzyme delivery. Loss of diffusion through plasma membrane perforin pores inhibits target cell delivery of granzyme B. (A) Indo-1-AM–labeled Jurkat cells were treated with recombinant perforin at 37°C for 30 minutes. Relative intracellular calcium concentrations were determined by the ratio of violet:blue (420:510 nm) wavelengths. Shown are the mean Indo-1 ratio over time. Arrow indicates the addition of recombinant perforin following establishment of a baseline reading. The shaded region indicates the increase in intracellular calcium, and “Restoration Phase” represents the decrease in intracellular calcium. Data are representative of 3 independent experiments. (B) Kinetics of repair: Jurkat cells were untreated (0 minutes) or treated with perforin at 37°C before shifting cell aliquots to 4°C at 1, 2, 3, 4, 5, 7, 10, 15, or 30 minutes after perforin treatment. The percentage of 7-AAD⁺ cells was then determined. Data represent the mean ± standard error (n = 3 independent experiments). The “Exclusion Phase” indicates the loss of 7-AAD⁺ cells. (C) Bid cleavage analysis: Jurkat cells were treated with perforin and incubated at 37°C before shifting cell aliquots to media alone (−Grz) or media containing granzyme B (62.5 nM) at 1, 2, 3, 4, 5, 7, 10, 15, and 30 minutes after perforin incubation, and then incubating for a further 10 minutes at 37°C. Whole cell lysates were prepared, and the formation of tBid was assessed by Bid immunoblot analysis. The migration of uncleaved Bid and truncated Bid (tBid) is marked on the right side of the panels. The top panel represents a short exposure, and the bottom panel is a long exposure from the same blot. Data are representative of 2 independent experiments. (D) Nuclear fragmentation (% PI+ sub-G1 nuclei): cells were treated with perforin and incubated at 37°C before shifting cell aliquots to media alone (Prf only) or media containing granzyme B (+GrzB; 15.6 nM) at 1, 2, 3, 4, 5, 7, 10, 15, and 30 minutes after perforin incubation, followed by incubation at 37°C/5% CO₂ for 3 hours. Shown is the quantification for perforin and granzyme B synergy or perforin-only treatments. Data represent the mean ± standard error (n = 3 independent experiments). (E) Phosphatidylserine exposure/7-AAD positivity: experiments were performed as in panel B, except the population of percentage of 7-AAD⁺ and/or AnnexinV⁺ cells was gated. Shown are the quantification for perforin and granzyme B synergy or perforin-only treatments. Data represent the mean ± standard error (n = 3 independent experiments). (F) Table shows the half-life (t1/2) of calcium restoration, 7-AAD exclusion (loss of 7-AAD⁺ cells), and perforin and granzyme synergy (loss of AnnexinV⁺ and/or 7-AAD⁺ cells) from their maxima. Data represent the mean ± standard error (n = 3 independent experiments).