Confocal microscope lsm880

LC3 puncta analysis was performed as we previously reported [34 (link)]. The Cells were seeded on coverslips in 24-well plates at a density of 5 × 10² cells per well in 1 ml of medium. After 24 h, cells were cotreated with HCQ and BKM120. After 48 h, cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 in PBS. Samples were then blocked in 5% donkey serum in the presence of 0.1% Triton X-100 and stained with the primary antibody γH2AX (Abcam, ab81299). After the cells were washed three times with PBS, the secondary antibody coupled to Alexa Fluor 488 was added and incubated for 1 h at room temperature. After being rinsed and washed three times with PBS, slides were mounted using VECTASHIELD mounting medium (Vector Laboratories) containing DAPI. Cells were then visualized with Zeiss Confocal microscope LSM880 for the presence of γH2AX puncta. The puncta was quantified using ImageJ.

Drosophila larvae were fixed in 8% paraformaldehyde (Electron Microscopy Science, Hatfield, U.S.A.) in PBS at room temperature for 30 min. After blocking for 2 h in 1% BSA in PBS/0.1% Triton-X 100, inverted larvae were incubated overnight in primary antibody [anti-5HT (Sigma-Aldrich AB125, 1:2000), anti-shroud antibody (gift from R.Niwa) (1:500), anti-Syt1 antibody (1:100, Developmental Studies Hybridoma Bank). Primary antibody staining was detected using Alexa Fluor 488 (Molecular Probes) goat-anti rabbit secondary antibodies (1:5000). DNA was visualized by staining with Hoechst 33258 (Invitrogen, 1:10,000). Brains with attached prothoracic glands were then dissected out and mounted on coverslips using mounting media (Vectashield). Images were captured using a Zeiss confocal microscope LSM 880 or a Zeiss Observer. Z1 microscope.

For the GFP-LC3 puncta assay, MCF7, MDA-MB-231 and MDA-MB-468 cells stably expressing GFP-LC3 were plated in six-well plates and treated with drug for 48 h. Following treatment, cells were washed with 1 × PBS, fixed with 4% paraformaldehyde for 10 min and mounted with Vectashield mounting media with DAPI. Cells were then visualized with Zeiss Confocal microscope LSM880 usign the 488 nm laser (GFP) for the presence of GFP-LC3 puncta. The puncta was quantified using ImgeJ.
For RFP-GFP-LC3 dual reporter assay, cells were transfected with ptf-LC3 vector and analysed as described previously24 (link). Briefly, ptfLC3 vector was tranfected using Lipofectamine 2000 as per manufacturer’s instructions. Cells were then treated with the drug for 48 h, washed with 1 × PBS, fixed briefly (for 10 min) with 4% paraformaldehyde and mounted with Vectashield moutning media. They were then visualized using with Zeiss Confocal microscope LSM880 using the 488 and 643 nm channels to image the GFP+ve and RFP+ve LC3 puncta respectively. The puncta was quantified using ImgeJ to obtained the number of autophagosomes (yellow −RFP+ GFP+) and autophagolysosomes (red −RFP+).

For co-localization analysis cells were fixed and stained for total PD-L1 and the protein of interest with primary antibody followed by Cy3 and Alexa488-labeled secondary antibodies. Cells were imaged on Zeiss Confocal microscope (LSM880) with Airyscan on a 40 × oil objective. Images were analyzed in MetaMorph software. Intensity threshold was manually determined for each colocalization marker and kept constant for a binary mask of the marker. The binary mask was overlayed with PD-L1 staining resulting in PD-L1 stain only in pixels positive for the marker proteins. For each cell, integrated intensity of PD-L1 staining in the original images as well as the overlayed images were calculated. Ratio of overlayed intensity over original intensity was used as “fraction under mask”. Unpaired t-test was performed on raw data.

Human fibroblasts were cultured in 24‐well chambers on poly‐L‐lysine‐coated coverslips overnight prior to treatments. Cells were treated with 30 μM agonist in DMSO (to a final DMSO concentration of 0.3%) for 48 h, and the filipin staining was initiated: Cells were washed twice with ice‐cold PBS, and fixed in 4% PFA for 30 min. Fixed cells were again washed with cold PBS, and unesterified cholesterol was visualized by filipin staining (PBS with 0.05 mg/mL filipin, Sigma‐Aldrich, and 10% FBS) for 2 h at room temperature in a dark humid chamber. Cells were subsequently washed with ice‐cold PBS twice, and nuclei stained using TO‐PRO‐3 (1:500, Invitrogen). Cells were washed twice and mounted on microscope slides overnight for imaging. Images were captured using a Zeiss Confocal Microscope (LSM 880), using a 40X oil objective, at 405 nm (filipin), 560 nm (mCherry), and 633 nm (TO‐PRO‐3). For data quantification, we calculated average filipin intensity per cell using Harmony High‐Content Imaging and Analysis Software (PerkinElmer).

Cells were plated on glass-bottom dishes prior to all experiments. After corresponding experimental treatments, cells were washed and fixed in 3.7% formaldehyde for 5 min. Cells were stained with primary and fluorescent secondary antibodies for proteins of interest on the plasma membrane without permeabilization. Saponin (0.25 mg/mL) was used to permeabilize the cells for total protein stain. Cell nuclei were labeled with Hoechst stain. For quantifications, images were acquired on an inverted microscope with a 20 × air objective or a 40 × oil objective (Leica Biosystems DMI6000-B). Images were analyzed on MetaMorph image processing software and the background corrected average intensity under each cell’s area was used to quantify protein amount. The values were normalized to the average of replicate coverslip dishes in control condition in each experiment to allow for comparison across experiments. Unpaired two tailed Student’s t test or paired ratio t-test were performed on unnormalized raw data, and one sample t test was used on normalized data respectively as indicated. For comparison of three and more groups, one-way ANOVA was performed on raw data with post-hoc Tuckey test where indicated. For PD-L1 stain images, Z stack of the cells was acquired using Zeiss Confocal microscope (LSM880) with Airyscan on a 40 × oil objective.

iPSC‐derived neurons were terminally matured in glass‐bottom, poly‐ornithine/laminin‐coated eight‐well chambers (ibidi) as previously described, using DAPT and 5‐FU for 7 days, and kept in culture for another week before imaging. iPSC‐derived neurons were treated with 0.3% DMSO or 30 μM TPC2‐A1‐P for 48 h prior to live‐cell imaging. LyTr‐DR was added at a dilution factor of 1:10,000 to the culture medium 30 min prior to confocal microscopy. The cells were transferred to a pre‐heated 37°C incubation chamber mounted onto a Zeiss Confocal microscope (LSM 880) and imaged using a 63 X water objective and an excitation wavelength of 633 nm (LyTr). Quantification of captured images was performed using the Fiji software. A mask was generated around the neuronal cell bodies, and the mean intensity was recorded.