DOI: 10.5281/zenodo.10479096
title: Confocal microscopy dataset: Effect of glutamine starvation and NVP-BEZ235 on mitophagy and lipid peroxidation

description: Short annotation

Confocal microscopy dataset: Effect of Glutamine starvation and autophagy inducer and PI3K/mTOR inhibitor NVP-BEZ235 on mitophagy (staining DAPI, mitotracker, p62, BNIP3, LC3, TSG101) and lipid peroxidation (Lipid Peroxidation kit), Accompanying the scientific paper: 

Bugajova, M., et al: Glutamine starvation alters the ATP production, oxidative stress, and abundance of mitochondrial RNAs in extracellular vesicles produced by cancer cells (submitted, DOI TBA)

Fixed Cells

First, the cells were seeded on 18 mm coverslips coated with 0.5% gelatin and cultured under standard conditions. After reaching 70% confluence, the cells were exposed to the desired conditions for 24 h. Prior to fixation, cells were incubated with 150 nM MitoTracker™ Red CMXRos (Thermo Fisher Scientific, Waltham, Ma, USA) for 45 min and washed three times with a pre-warmed medium. Then, the cells were fixed in a pre-warmed fix solution containing 4% paraformaldehyde, 3% sucrose, PBS, and water for 20 min at 37 °C and washed three times with PBS. Right after, the cells were permeabilized using 0,2% Triton X-100 for 10 min. Permeabilization was followed by the blocking of non-specific Ab-blocking sites in PBS-BSA 0,1% for 60 min. Such prepared cells were incubated with primary antibodies (SQSTM1/p62 1:200, MAPLC3 1:200, BNIP3 1:200, TSG101 1:200) for 90 min at 37°C. Then, the cells were washed three times with PBS 0.05% Tween 20 (PBS-T) and incubated with compatible secondary antibodies (Alexa Fluor® 488, 1:750; Alexa Fluor® 647, 1:750) for 60 min in the dark at RT. After secondary antibody incubation, the cells were washed two times with PBS-T and then incubated with 7 μl/50 ml DAPI. Prior to the mounting, cells were washed two times with PBS-T and once with PBS. Coverslips were mounted on glass slides using ProLong™ Gold Antifade Mountant (Thermo Fisher Scientific, Waltham, Ma, USA).

Live cells

For live cell imaging, cells were cultured in Ibidi µ-Slide VI 0.4 (ibiTreat, Cat. No. 80606). After reaching 70% confluence, cells were washed twice with PBS and exposed to the treatment for 24 h. For lipid-peroxidation assay Image-iTTM, a Lipid Peroxidation kit (Thermo Scientific) was used, following the manufacturer's protocol. The Image-iTTM kit enables the detection of lipid peroxidation in live cells through oxidation of BODIPY™ 581/591 C11 reagent. 

Acquisition

Confocal microscopy images of fixed cells were acquired using Laser scanning confocal microscope Zeiss LSM 880 with AiryscanFast module (Carl Zeiss Inc.) using a Plan-Apochromat 63× / 1.40 OIL objective. DAPI was excited with 405 nm solid state laser, and emitted light was detected at 433 nm. Alexa Fluor™ 488 was excited with 488 nm Argon laser, and emitted light was detected at 516 nm. MitoTracker™ Red CMXRos was excited with 561 nm solid state laser, and emitted light was detected at 579 nm. Alexa Fluor™ 647 was excited with HeNe 633 nm laser and emitted light was detected at 654 nm. Live cells images were acquired using a Plan-Apochromat C-Apochromat 63× / 1.20 W objective. Oxidized Bodipy was excited with 488 nm Argon laser, and emitted light was detected at 541 nm and reduced Bodipy was excited with 561 nm solid state laser, and emitted light was detected at 640 nm. AiryScan images were acquired using 1.8× magnification and processed using Zen Black software (Carl Zeiss Inc.). Colocalization data were evaluated using Coloc tool using the Zen Blue Software (Carl Zeiss Inc.).

File description

Files created on LSM880 are stored in original CZI files, files acquired with airy scan mode are saved after airyscan processing. Files are located in three separed ZIP files corresponding to staining setup:



lipid_peroxidation_oxBODIPY_BODIPY_Confocal_live_cells_63x (staining of live cells)

mitophagy_DAPI_LC3_Mitotracker_BNIP_Confocal_Airyscan_fixed_cells_63x (staining of fixed cells, first set of antibodies for mitophagy)

mitophagy_DAPI_p62_mitotracker_TSG101_Confocal_Airyscan_fixed_cells_63x (staining of fixed cells, second set of antibodies for mitophagy)


for file naming, the CTRL is for control, untreated cells, for BEZ for NVP-BEZ235 and starv for Gln starvation. For experimental detail see methods section of the article mentioned above.
description: We acknowledge the core facility CELLIM supported by MEYS CR (LM2018129 Czech-BioImaging).