Figure 2.
Figure 2. Exocytosis of CGs is unchanged in Flower-deficient CTLs. (A) FACS-based degranulation assay with CTLs derived from WT and Flower-deficient mice by using CD107-PE conjugated antibody. Cells were stimulated with 10 µg anti-CD3 antibody and compared with unstimulated (constitutive) cells. Cells in the absence of CD107-PE antibody served as controls. One representative experiment of five (see B) is shown. (B) Statistical analysis of the average percentage of cells exhibiting CD107a fluorescence at the plasma membrane after stimulation (N = 5; n = 10). (C) Representative TIRFM serial images showing the fusion of a CG (arrow). Bar, 2 µm. (D and E) CG fusion analysis. CTLs from WT and Flower-deficient mice were transfected with syb2-mRFP as a CG marker. Cells were plated on anti-CD3–coated coverslips and perfused with 1 mM calcium buffer for TIRFM recording. Percentage of cells that show CG fusion (D) and the number of fusion events per cell (E) are shown. (F) CG fusion latency. CTLs were transfected with syb2-mRFP as a CG marker. CG fusion events were triggered by seeding CTLs on anti-CD3 antibody-coated glass coverslips. The fusion latency expresses the duration from the first granule appearing in the TIRFM plane to the fusion of individual CGs. Unpaired Student´s t test: ***, P < 0.001. (G) Polarization latency of recycling endosomes (REs). CTLs were transfected with rab11-mCherry as an RE marker and recorded in 1 mM calcium buffer by TIRFM. The polarization latency of the duration from a CTL attaching to the anti-CD3 coated coverslip to the first RE appearing in the TIRFM plane. (H and I) Ratiometric imaging of cytosolic calcium (Fura F340/F380) in CTLs derived from WT (black) and Flower-deficient (red) mice seeded on poly-l-ornithine (H)– and anti-CD3 (I)–coated coverslips. (J–M) Quantification of data shown in panels H and I. Basic cytosolic calcium levels (Fura F340/F380) (J), amplitude of the anti-CD3–mediated cytosolic calcium increase (K), delay of the calcium increase (L), as well as the percentage of cells responding with an increase of cytosolic calcium while seeded on poly-l-ornithine and anti-CD3–coated coverslips (M) were not significantly different in CTLs derived from WT and Flower-deficient mice. (N) Ratiometric imaging of cytosolic calcium (Fura F340/F380) in CTLs derived from WT (black) and Flower-deficient (red) mice seeded on anti-CD3–coated coverslips after the addition of 10 mM extracellular calcium. Data in B, D, E, and H–N are given as mean ± SEM, and the number of experiments (x) and cells (n) is shown in brackets (x/n). Two-tailed unpaired Student´s t tests were performed.

Exocytosis of CGs is unchanged in Flower-deficient CTLs. (A) FACS-based degranulation assay with CTLs derived from WT and Flower-deficient mice by using CD107-PE conjugated antibody. Cells were stimulated with 10 µg anti-CD3 antibody and compared with unstimulated (constitutive) cells. Cells in the absence of CD107-PE antibody served as controls. One representative experiment of five (see B) is shown. (B) Statistical analysis of the average percentage of cells exhibiting CD107a fluorescence at the plasma membrane after stimulation (N = 5; n = 10). (C) Representative TIRFM serial images showing the fusion of a CG (arrow). Bar, 2 µm. (D and E) CG fusion analysis. CTLs from WT and Flower-deficient mice were transfected with syb2-mRFP as a CG marker. Cells were plated on anti-CD3–coated coverslips and perfused with 1 mM calcium buffer for TIRFM recording. Percentage of cells that show CG fusion (D) and the number of fusion events per cell (E) are shown. (F) CG fusion latency. CTLs were transfected with syb2-mRFP as a CG marker. CG fusion events were triggered by seeding CTLs on anti-CD3 antibody-coated glass coverslips. The fusion latency expresses the duration from the first granule appearing in the TIRFM plane to the fusion of individual CGs. Unpaired Student´s t test: ***, P < 0.001. (G) Polarization latency of recycling endosomes (REs). CTLs were transfected with rab11-mCherry as an RE marker and recorded in 1 mM calcium buffer by TIRFM. The polarization latency of the duration from a CTL attaching to the anti-CD3 coated coverslip to the first RE appearing in the TIRFM plane. (H and I) Ratiometric imaging of cytosolic calcium (Fura F340/F380) in CTLs derived from WT (black) and Flower-deficient (red) mice seeded on poly-l-ornithine (H)– and anti-CD3 (I)–coated coverslips. (J–M) Quantification of data shown in panels H and I. Basic cytosolic calcium levels (Fura F340/F380) (J), amplitude of the anti-CD3–mediated cytosolic calcium increase (K), delay of the calcium increase (L), as well as the percentage of cells responding with an increase of cytosolic calcium while seeded on poly-l-ornithine and anti-CD3–coated coverslips (M) were not significantly different in CTLs derived from WT and Flower-deficient mice. (N) Ratiometric imaging of cytosolic calcium (Fura F340/F380) in CTLs derived from WT (black) and Flower-deficient (red) mice seeded on anti-CD3–coated coverslips after the addition of 10 mM extracellular calcium. Data in B, D, E, and H–N are given as mean ± SEM, and the number of experiments (x) and cells (n) is shown in brackets (x/n). Two-tailed unpaired Student´s t tests were performed.

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