Plan apochromat 20x

SMG morphological evaluation was performed using a digital inverted fluorescence microscope (Nikon, Tokyo, Japan; Ti) equipped with a digital camera (Nikon, DS-Ri2) and a CFI Plan Fluor 4x objective (Nikon) or JuLI Br live cell movie analyzer (NanoEnTek, Seoul, Republic of Korea). Immunofluorescence images were taken by confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany;LSM700) equipped with Plan-Apochromat 10x, Plan-Apochromat 20x, and C-Apochromat 40x objectives (Carl Zeiss) and with 405, 488, and 555 nm wavelength excitation lasers. Live imaging of epithelial rudiments of SMG and SMG-C6 cells were conducted through a confocal microscope (Carl Zeiss) with a customized live cell chamber (Live Cell Instruments, Seoul, Republic of Korea) that maintained 5% CO₂ and 37 °C conditions. To visualize peripheral cell movement (Fig. 4I,J), the epithelial rudiments of SMGs were briefly stained with 1 μg/ml Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA; H3570) –culture media solution for 1 h. After staining, cells were washed with culture medium two times.

hMDMs at 900000 cells/well in 27 mm Nunc glass bottom dishes (Thermofisher) were washed twice with imaging media (HBSS containing 5mM glucose, 20 mM HEPES and 1% BSA) and stained with 500 nM MitoTracker Red CMXRos for 15 min. Cells were washed twice and visualized on a Zeiss LSM880 confocal microscope before data capture. Cells were then treated with sublytic MAC, anti-C7 control or 5 μM ionomycin for 15 min in imaging media without BSA, and visualized on the above system with a Zeiss Plan-Apochromat 20x or 63x objective. For 20X, 9 images were taken for each condition and donor (3 x 3 tile), for 63X, 5 images per condition and donor were taken. Mitochondrial dynamics were quantified using the Yen threshold in the semi-automated analysis macro tool MiNA, used with Fiji/ImageJ software (46 (link)) (4 cells per condition per donor analyzed). The mitochondrial branch length mean values were exported, averaged and used as a measure for mitochondrial network morphology.

Microscope images of fixed Drosophila follicles were obtained using LAS AS SPE Core software on a Leica TCS SPE mounted on a Leica DM2500 using an ACS APO 20 x/0.60 IMM CORR -/D objective (Leica Microsystems, Buffalo Grove, IL) or using Zen software on a Zeiss 700 LSM mounted on an Axio Observer.Z1 using a Plan‐Apochromat 20 x/0.8 working distance (WD) = 0.55 M27 or a EC-Plan-Neo-Fluar 40 x/1.3 oil objective (Carl Zeiss Microscopy, Thornwood, NY). Maximum projections (two to four confocal slices), merged images, rotations, and cropping were performed using ImageJ software (Abramoff et al., 2004 ). S9 follicles were identified during fixed imaging by the size of the follicle (~150–250 μm), the position and morphology of the outer follicle cells, and presence of a border cell cluster. The beginning of S10 was defined as when the anterior most outer follicle cells reached the nurse cell-oocyte boundary and flattened.

MLCK-FRET plasmid is a kind gift from Dr. James T. Stull (University of Texas Southwestern Medical Center). The MLCK-FRET plasmid is a calmodulin-binding based sensor, where calmodulin binding sequence is flanked with eCFP and eYFP and exhibits decreased FRET upon binding with calmodulin^{19 (link),38 (link)}. Cells expressing MLCK-FRET were imaged using Zeiss LSM 880 (Carl Zeiss, Jena, Germany), at 37 °C incubator, fitted with Plan-Apochromat 20 x, equipped with 458 nm and 514 nm Argon ion laser lines for excitation of eCFP and eYFP respectively. Incident excitation light was split using an MBS 458 nm/514 nm beam splitter and collected on a 32-spectral array GaAsp detector. The fluorescence emission was collected from 463–520 nm (ECFP), 544–620 nm (FRET channel and eYFP channel). Intensity based ratiometric FRET were obtained using custom-written scripts in ImageJ and MATLAB. Since MLCK-FRET is a single-chain construct, decrease in FRET, and increase in MLCK binding to calmodulin, was expressed as the ratio of emission intensity at 520 nm/emission intensity at 510 nm normalized at the basal levels.