Clsm 710 spectral confocal laser scanning microscope

MDA-MB-231, BT474 and MCF10A cells were seeded at 1 × 10⁵ cells/well in 8-well Lab-Tek II Chamber slides. Cells were allowed to adhere for several days and were treated with a specified dosage of Alexa Fluor^®647-PAA-P85-SMNPs. After 24 h, the cells were washed and then exposed to an AC magnetic field (50 Hz, 50 kA/m) using the pulsed exposure regime for a total of 30 min (i.e., 10 min on, 5 min off, for a total of 30 min on). Twenty four hours post exposure, the cells were treated with 100 nM Lysotracker™ Green (λ_ex/λ_em = 504/511 nm) for 1 h and Hoechst 33342 nuclear stain for 15 min. Cells were washed 3X with PBS and kept in complete media for imaging. Live cell images were acquired using a Zeiss CLSM 710 Spectral Confocal Laser Scanning Microscope with the 63X/1.4 Oil Plan Apo lens.

pRBC^DDAO and pRBC^Violet were stained with 1× DNA dye SYBR Green I, fixed with 1% paraformaldehyde and 0.0075% glutaraldehyde, and then viewed by oil immersion with a 63X/1.4 numerical aperture Oil Plan Apo objective lens on a Zeiss CLSM 710 Spectral Confocal Laser Scanning Microscope. Images were captured with Zeiss ZEN 2011 software.

HL-1 cardiomyocytes (2.5 × 10⁵/well/50% confluent) plated on Gelatin/Fibronectin were co-transfected with Flag-PPARγ1 and HA-MuRF2 using Lipofectamine LTX & PLUS (Invitrogen, lot#1397274) according to the manufacturer’s instructions. The ratios of LTX/DNA and PLUS/DNA (μl/μg) both were 2:1. Equal amounts of DNA were transfected by adjusting with empty vectors. After 48 h of transfection, the cells were fixed with 4% paraformaldehyde and blocked in 5% goat serum with 0.2% TritonX-100 at room temperature for 1 h. Cells were incubated with Rb anti-Flag (Sigma F7425, 1:100, 4°C, overnight or Ms anti-HA (Sigma H9658, 1:100, 4°C, overnight). Cells were washed and incubated with anti-Ms 488 to detect HA-MuRF2 (Invitrogen, 1:1,000) or anti Rb 568 (Invitrogen, 1:1,000) for 1 h at room temperature. The membranes were cut into 1 × 1 cm sections and mounted to glass slides with Fluoro-Gel Anti-fade mounting medium with DAPI (EMS, Hatfield, PA Cat. #17983-20) and analyzed by fluorescent confocal microscopy using a Zeiss CLSM 710 Spectral Confocal Laser Scanning Microscope.

Confocal images were captured on a Zeiss CLSM 710 Spectral Confocal Laser Scanning Microscope, using objective lenses of either 20× or 40× magnifications (numerical apertures of 0.80 and 0.95, respectively). Preprocessing of the raw images, thresholding and masking was performed for each confocal slice as described in the previous section. eGFP was quantified relative to DsRed in each slice as described in the prior section and then averaged over all slices possessing colonoids to yield the percentage colonoid volume positive for eGFP. To quantify the Muc2- and ChgA-expression or EdU-staining regions in each image slice, the number of pixels positive for these markers was divided by the number of pixels positive for Hoechst 33342. The average ratio for every slice in a sample was then calculated to yield the average volume of sample positive for Muc2, ChgA, or EdU relative to that positive for Hoechst 33342.