4T1 cells were seeded onto glass coverslips in 6-well plates and incubated overnight in RPMI-1640 media at 37°C. The 20 μL solutions with different concentrations of the 9T-PUNNC (12.5, 25, 50, and 100 μg mL-1) were then added to the culture media and the cells were continued to incubate for 12 h. After 5 min of NIR-II irradiation, the cells were co-stained with calcein-AM (green, live cells) and PI (red, dead cells) for 15 minutes. The confocal fluorescent images were acquired using a Leica TCS SP8X laser scanning microscope (Leica Microsystems GmbH, Germany). Two different excitation wavelengths were used, including 490 and 545 nm.
Tcs sp8 x laser scanning microscope
Manufactured by Leica
Sourced in Germany
The Leica TCS SP8 X is a laser scanning microscope designed for high-resolution imaging. It features a fully-motorized stage and objective turret, enabling automated acquisition and analysis of samples. The microscope is equipped with multiple laser lines, allowing for flexible excitation of a wide range of fluorophores. The system's advanced optics and detectors provide high-quality, detailed imaging capabilities.
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Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Evos floid cell imaging station, by Thermo Fisher Scientific (1 mentions)
Hc pl apo cs2 63 1, by Leica (1 mentions)
Mitotracker deep red, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated anti mouse antibody, by Jackson ImmunoResearch (1 mentions)
Anti lc3b, by Cell Signaling Technology (1 mentions)
Vectashield dapi, by Vector Laboratories (1 mentions)
Fluorescein labelled avidin d, by Vector Laboratories (1 mentions)
Mowiol 4 88 antifade mounting medium, by Merck Group (1 mentions)
Biotinylated anti avidin d, by Vector Laboratories (1 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (1 mentions)
7 protocols using tcs sp8 x laser scanning microscope
1
Photothermal Cytotoxicity of 9T-PUNNC
2
Intracellular ROS Detection in 4T1 Cells
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The ROS probe DCFH-DA was used to detect the ability of intracellular ROS generation 59 (link). 4T1 cells were cultured in a six-well plate for 24 h. And then, 1 mL 9T-PUNNC solution (100 μg mL-1) was added into 4T1 cells and further cultured for 3 h. The DCFH-DA was added for 20 min and washed with PBS several times. Finally, the fluorescence image was obtained using a Leica TCS SP8X laser scanning microscope (Leica Microsystems GmbH, Germany) with λex=488 nm.
3
Immunocytochemistry Staining and Microscopy
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Immunocytochemistry staining was performed as described previously [16, (link)17] (link). MitoTracker Deep Red (100 nM; Thermo Fisher Scientific, Waltham, MA, USA) was added after hyperoxic treatment and cells were incubated for 20 min at 37°C. Primary and secondary antibodies used for immunocytochemistry in this study are listed in Table 2. Confocal imaging was performed using Leica TCS SP8 X laser scanning microscope (Leica Microsystems, Wetzlar, Germany), equipped with an HC PL APO CS2 63/1.40 oil immersion objective and a white light laser. The excitation wavelengths and emission detection ranges used were 405 nm and 412-460 nm for DAPI, 488 nm and 495-550 nm for Alexa488, 594 nm and 601-644 nm for Alexa594, and 644 nm and 651-700 nm for MitoTracker Deep Red, respectively. For cell morphology analysis on EVOS Floid Cell Imaging Station (Thermo Fisher Scientific, USA), live cells in the medium were stained with NAO (1,5 µM) and Hoechst 33342 (5 µg/ml; Thermo Fisher Scientific, USA) for 5 min, washed 2x with 1x PBS and maintained in 1x PBS+Mg/Ca.
4
Metformin Induces Autophagy in FaDu Cells
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FaDu cells were grown on glass coverslips in 6-well plates and treated with 0, 5, or 10 mM metformin for 48 h. The treated cells were fixed with 4% formaldehyde for 10 min and permeabilized with 0.1% Triton X-100 in PBS at 4 °C for 10 min. The samples were then blocked with 3% bovine serum albumin (BSA) in PBS at 37 °C for 30 min, then stained with anti-LC3B (Cell Signaling Technology no. 3868; diluted 1:200) and Alexa Fluor 488-conjugated anti-mouse antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA; diluted 1:400). 4′,6-diamidino-2-phenylindole (DAPI) (0.2 μg/mL) was used for nuclear staining. The stained cells were then analyzed under a confocal microscopy (Leica TCS SP8X laser-scanning microscope using a 63 × 1.4 numerical aperture objective) [56 (link)].
5
Quantifying Autophagic Flux in Cells
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Cells expressing mRFP‐GFP‐LC3 were grown on glass coverslips and were incubated in full RPMI medium or starved in Earle's balanced salt solution for 4 h. After treatment, cells were fixed in PFA 2% and mounted in Vectashield/DAPI (Vector Laboratories). Images were blindly acquired on a Leica TCS SP8 X laser scanning microscope. Processing and analysis of images took place with ImageJ/Fiji. ROIs (individual cells) from one imaging field were manually selected with the ROI manager function. Binary images were created from individual green fluorescent protein (GFP) and red fluorescent protein (RFP) channels by subjecting them to fixed thresholding settings, followed by application of watershed segmentation. Colocalization images (yellow puncta) were generated with Image Calculator from RFP and GFP segmented images using the intersection (AND operator), followed by analysis of yellow puncta (autophagosome) number with the analyze particles function. Relative mean yellow puncta per cell were determined for 60−70 cells per condition.
6
Fluorescence In Situ Hybridization (FISH) Protocol
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FISH experiments were performed according to the protocol described in Pérez-García et al. [42 (link)] with the adaptation in pepsin digestion (5 min at 37 °C). Prior to application, probes were denatured at 80 °C for 8 min and cooled on ice for 2 min. Detection was performed using fluorescein-labelled avidin D (Vectashield) and biotinylated anti-avidin D (Vectashield). Slides were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI; 100 ng/mL) and mounted with Mowiol 4-88 antifade mounting medium (Sigma-Aldrich, St. Louis, MO, USA). Slide visualization was performed using a Nikon Eclipse-800 fluorescent microscope and a Leica TCS SP8 X laser scanning microscope.
7
Immunofluorescence Imaging Protocol for Cells
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Cells were plated onto glass coverslips and following indicated treatment/incubation, cells were washed in 1xPBS and fixed using 4% PFA (10 mins, RT) or modified MEM-Fix (4% formaldehyde, 0.25-0.5% glutaraldehyde, 0.1M Sorenson's phosphate buffer, pH7.4) (Bodeen et al., 2017; (link)Rogers and Scholpp, 2021) (link) for 7 mins at 4 o C. Cells were then incubated in permeabilisation solution (0.1% Triton-X-100, 5% serum, 0.1M glycine in 1xPBS) for 1hr at RT. Primary antibodies were diluted in incubation buffer (0.1% Tween20, 5% serum in 1xPBS) and coverslips incubated in 50 µl spots on parafilm overnight at 4 o C. Coverslips were then washed with 1xPBS 3x for 5 mins before incubation in 50 µl (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted January 8, 2022. ; https://doi.org/10.1101/2022.01.07.475396 doi: bioRxiv preprint 16 spots of secondary antibodies diluted in incubation buffer for 1hr at RT. Coverslips were then washed 3x for 5 mins with 1xPBS before mounting onto glass slides using ProLong Diamond anti-fade mountant (Invitrogen) and left to dry for 24hrs before imaging. Confocal microscopy for immunofluorescent antibody imaging was performed on an inverted Leica TCS SP8 X laser-scanning microscope using the 63x water objectives.
The copyright holder for this preprint this version posted January 8, 2022. ; https://doi.org/10.1101/2022.01.07.475396 doi: bioRxiv preprint 16 spots of secondary antibodies diluted in incubation buffer for 1hr at RT. Coverslips were then washed 3x for 5 mins with 1xPBS before mounting onto glass slides using ProLong Diamond anti-fade mountant (Invitrogen) and left to dry for 24hrs before imaging. Confocal microscopy for immunofluorescent antibody imaging was performed on an inverted Leica TCS SP8 X laser-scanning microscope using the 63x water objectives.
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