![Figure 4. Functional assays and calcium levels in NK cells derived from mouse spleen. (A) Killing of YAC-1 cells by NK cells prepared from control Npc1+/+ or affected Npc1−/− mouse splenocytes. YAC-1 cells were identified as carboxyfluorescein diacetate succinimidyl ester (CFSE)bright, and specific killing was determined using the following equation at the effector to target ratio indicated: (dead YAC-1 cells with NK cells) − (dead YAC-1 cells with no NK cells). (B-D) NK cells were identified as NKp46+CD3−, doublet events were excluded by FSC-H vs FSC-A gating, and a viability dye was included to exclude dead cells from the analysis. (B) Degranulation of NK cells from control Npc1+/+ and Npc1−/− animals in response to stimuli in the presence of monensin was calculated according to the following equation: (% of CD107a+ NK cells with stimuli) − (% of CD107a+ unstimulated NK cells). (C) Intracellular IFN-γ production by NK cells from control Npc1+/+ and Npc1−/− animals in response to stimuli was calculated according to the following equation: (% of IFN-γ+ NK cells with stimuli) − (% of IFN-γ+ unstimulated NK cells). (D) Representative traces showing intracellular [Ca2+] changes monitored in single fluo-4–loaded NK cell, normalized to initial fluorescence (F/F0). Lysosomal calcium content was assessed upon addition of 50 μM GPN which lyses cathepsin-containing acidic intracellular calcium stores. The addition of ionomycin at the end of each experiment was used to confirm viability of the cells. (E) Maximal peak fluorescence changes were determined as the difference between basal and the maximum fluorescence, Δ(F/F0), upon addition of 50 μM GPN. Data are presented as the mean ± SEM, n = 3 animals for each group; 158 Npc1+/+ cells and 125 Npc1−/− cells. Clear bars represent control Npc1+/+ animals and solid bars represent affected Npc1−/− animals. (F) Degranulation of NK cells from control Npc1+/+ and Npc1−/− animals in response to PMA/iono in the presence or absence of monensin was determined as described for panel B. Data are presented as mean ± SEM, n = 4 to 6 animals. *P < .05, **P < .01, and ***P < .001, calculated by an unpaired t test using GraphPad Prism v4.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/123/1/10.1182_blood-2013-03-488692/4/51f4.jpeg?Expires=1735509542&Signature=VkH-O5onLsZmrw~kjMtIBmm1tqLgj6c8Zd9TVaeB2AFY3cxsVmqQzoo-34oMqO-6bA743BjrWoz-mXo-zq26wWJforymBsjAUQ-TxBnod3Ba7aaQdviss2sTWFE-7BrJTZWQD~F7hH8hJsh2qE1XHj8emFHks8JbG3H-8w6R1v2NOhz1L-gAArnvqChShLMkaTuX68G5~~Y-006c-4sKoPxYU8e4G-qFbKZ5QSyh3zrJk11Xsvjve9HQRiJZTS5Fo9uPzICxWgbeQAAmay0ZwpoEpBRxqS8t2B6jUVPFZ4GKUrWB4Cal7uuM9RZxqVWkdd~4ksqSBcb6vaojHyOe0w__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Functional assays and calcium levels in NK cells derived from mouse spleen. (A) Killing of YAC-1 cells by NK cells prepared from control Npc1+/+ or affected Npc1−/− mouse splenocytes. YAC-1 cells were identified as carboxyfluorescein diacetate succinimidyl ester (CFSE)bright, and specific killing was determined using the following equation at the effector to target ratio indicated: (dead YAC-1 cells with NK cells) − (dead YAC-1 cells with no NK cells). (B-D) NK cells were identified as NKp46+CD3−, doublet events were excluded by FSC-H vs FSC-A gating, and a viability dye was included to exclude dead cells from the analysis. (B) Degranulation of NK cells from control Npc1+/+ and Npc1−/− animals in response to stimuli in the presence of monensin was calculated according to the following equation: (% of CD107a+ NK cells with stimuli) − (% of CD107a+ unstimulated NK cells). (C) Intracellular IFN-γ production by NK cells from control Npc1+/+ and Npc1−/− animals in response to stimuli was calculated according to the following equation: (% of IFN-γ+ NK cells with stimuli) − (% of IFN-γ+ unstimulated NK cells). (D) Representative traces showing intracellular [Ca2+] changes monitored in single fluo-4–loaded NK cell, normalized to initial fluorescence (F/F0). Lysosomal calcium content was assessed upon addition of 50 μM GPN which lyses cathepsin-containing acidic intracellular calcium stores. The addition of ionomycin at the end of each experiment was used to confirm viability of the cells. (E) Maximal peak fluorescence changes were determined as the difference between basal and the maximum fluorescence, Δ(F/F0), upon addition of 50 μM GPN. Data are presented as the mean ± SEM, n = 3 animals for each group; 158 Npc1+/+ cells and 125 Npc1−/− cells. Clear bars represent control Npc1+/+ animals and solid bars represent affected Npc1−/− animals. (F) Degranulation of NK cells from control Npc1+/+ and Npc1−/− animals in response to PMA/iono in the presence or absence of monensin was determined as described for panel B. Data are presented as mean ± SEM, n = 4 to 6 animals. *P < .05, **P < .01, and ***P < .001, calculated by an unpaired t test using GraphPad Prism v4.
