LV-transduced and non-transduced cells were washed twice and 1000 cells at 1000 cells/100 µL, unless stated otherwise in the figure legend, were seeded in a 96-well Nunclon Sphera ultralow-affinity plate and cultured in their ATCC-recommended media to allow for spheroid formation verified by ZOE fluorescent Cell Imager (Bio-Rad). NK-92mi were labeled with Cell Trace Far-Red (Thermo Fisher Scientific) and 1000 cells at 1000 cells/100 µL, unless stated otherwise in figure legend, were added to each well after 48 h. Daily visual confirmation of spheroid formation, measurement of diameter using ImageJ software36 (link), and NK-92mi infiltration were documented via a ZOE fluorescent Cell Imager (Bio-Rad) recorded for 96 h.
Zoe fluorescent cell imager
Manufactured by Bio-Rad
Sourced in United States, Germany, Italy, Spain, Singapore, Australia
The ZOE Fluorescent Cell Imager is a compact, easy-to-use fluorescence imaging system designed for visualization and analysis of cells. It provides high-quality images and supports a range of fluorescent dyes and proteins.
Automatically generated - may contain errors
Lab products found in correlation
Rhodamine phalloidin, by Thermo Fisher Scientific (16 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (11 mentions)
Triton x 100, by Merck Group (10 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (9 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (6 mentions)
Hoechst 33342, by Thermo Fisher Scientific (6 mentions)
Cellevent caspase 3 7 green detection reagent, by Thermo Fisher Scientific (6 mentions)
Anti ahr, by Merck Group (5 mentions)
Texas red goat anti rabbit, by Thermo Fisher Scientific (5 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (5 mentions)
Bovine serum albumin (bsa), by Merck Group (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Cy3 conjugated goat anti rabbit igg secondary antibody, by Abcam (4 mentions)
Non fat dry milk, by Bio-Rad (4 mentions)
Paraformaldehyde (pfa), by Merck Group (4 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (4 mentions)
6 well plate, by Corning (3 mentions)
Pbs tween, by Merck Group (3 mentions)
Tc20 automated cell counter, by Bio-Rad (3 mentions)
Effectene, by Qiagen (3 mentions)
346 protocols using zoe fluorescent cell imager
1
Spheroid Formation and NK-92mi Infiltration
2
Autophagy modulation in ARPE-19 cells
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ARPE-19 cells were transiently co-transfected with ubiquitin-RFP and LC3-GFP plasmids using Lipofectamine® 2000 (Invitrogen, 24 hours) in a 24 well plate, and treated with 10% CSE, cysteamine (250 μM, Sigma), or fisetin (40 μM, Sigma) for 12 hours. Images were captured using the ZOE™Fluorescent Cell Imager (Bio-Rad). For the autophagy flux assay, ARPE-19 cells were transiently transfected with the Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B (Thermo Fisher) plasmid in a 24 well plate for 12 hours as described recently [24 (link), 25 (link)], and then subsequently treated with 10% CSE and/or cysteamine (250 μM, Sigma), for 12 hours. Images were captured using the ZOE™Fluorescent Cell Imager (Bio-Rad).
3
Synergistic Phototherapy via Liposomal Nanoparticles
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Cell viability was evaluated on CT26 cells (3 × 104 cells/well) with DMEM and was incubated overnight. Next, the cells were incubated with predetermined concentrations of ICG, PTX, LIL NPs and LIL-PTX NPs for 24 h. BSO (20 µM) was pretreated for 30 min prior to nanoparticle treatment. For laser treatment groups, the laser (808 nm, 2 W/cm2, 10 min) was irradiated, and the cells were replaced with fresh media followed by 24 h incubation. Thermal elevation in CT26 cells was performed by collecting the cell pellets post treatment with LIL-PTX NPs, followed by laser irradiation (808 nm, 2 W/cm2, 10 min). The temperature rise was recorded by a thermal camer (Avio IR camera/Thermometer, Japan). For the cytotoxicity evaluation, MTS (20 µL) reagent was incubated for 3 h, and the absorbance was recorded using a Spark 10 M multimode microplate reader (Tecan Trading AG, Switzerland) at 490 nm. The live/dead assay was carried out on LIL-PTX-treated cells with a laser (808 nm, 2 W/cm2, 10 min) by FDA and PI treatments according to the manufacturer's protocol and were imaged by a ZOE fluorescent cell imager (Bio-Rad, USA). The trypan blue assay was performed by adding 100 µL trypan blue (0.1 wt %), followed by PBS wash and was visualized by using a ZOE fluorescent cell imager (Bio-Rad, USA).
4
Fluorescent Cell Intake and Decline Assay
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For the intake ability test, 2 × 104 cells were seeded in 96 black wells for 1 day and treated with 100 μM FITC (Millipore-Sigma) for 5, 10, 30, and 60 min. Subsequently, the cells were washed with 1× PBS thrice and detected using the Fluorescent Cell Imager (ZOE; Bio-Rad, Hercules, CA, USA).
For the declining test, 2 × 104 cells were seeded in 96 black wells for 1 day and treated with 100 μM FITC for 60 min. Subsequently, the cells were washed with 1x PBS thrice and detected using the Fluorescent Cell Imager (ZOE; Bio-Rad, Hercules, CA, USA) at 0, 0.5, 1, and 3 h after FITC treatment.
All images were further analyzed using ImageJ for quantification. After acquiring the pixel number from GFP, the pixel number of the untreated sample was excluded from all data to reduce the noise from the background.
For the declining test, 2 × 104 cells were seeded in 96 black wells for 1 day and treated with 100 μM FITC for 60 min. Subsequently, the cells were washed with 1x PBS thrice and detected using the Fluorescent Cell Imager (ZOE; Bio-Rad, Hercules, CA, USA) at 0, 0.5, 1, and 3 h after FITC treatment.
All images were further analyzed using ImageJ for quantification. After acquiring the pixel number from GFP, the pixel number of the untreated sample was excluded from all data to reduce the noise from the background.
5
Live-Dead Cells Quantification
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Cells (1 × 103) were cultured in a complete medium. After 24 h of starvation, serum-free medium or serum-free medium containing different experimental treatments were added. After 48 h, the Cyto3DTM Live–Dead Assay Kit (TheWell Bioscience, Inc., North Brunswick, NJ, USA) was used to determine the live/dead nucleated cells using a dual-fluorescence system of acridine orange (AO) and propidium iodide (PI), both nuclear staining (nucleic acid binding) dyes. All live nucleated cells (green), and all dead nucleated cells (red) were counted. Several images were obtained using a Bio-Rad ZOE fluorescent cell imager (Bio-Rad Laboratories). Quantification analyses were performed using ImageJ (https://imagej.net/software/fiji ), a Java-based freeware, analyzing the mean gray value of the green and red channels separately, and then calculating the green/red ratio.
6
3D Spheroid Invasion Assay for Metastasis
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Cultrex 3-D spheroid cell invasion assay (Cultrex #3500-096-K) was
employed for three-dimensional (3D) invasion assay, following the
manufacturer’s instructions. Spheroids of A375 transfected cells were
generated as previously reported by plating 500 cells for 48 h in 3D culture
qualified 96-well plate [36 (link)]. Then, spheroids were embedded into the Cultrex spheroid
invasion matrix. After 1 h at 37 °C, SFM with or without broad-spectrum
matrix metalloprotease inhibitor, Ilomastat/GM6001 (Cat# GM6001;
Millipore, Burlington, Massachusetts, USA) or uPAR inhibitor, M25 peptide, was
added to spheroids wells. Plates were incubated for 7 days, and spheroids were
photographed every 24 h through the use of Bio-Rad ZOE fluorescent cell imager
(Bio-Rad). ImageJ software (https://imagej.nih.gov/ij/ ) was used to measure invasion area.
Captured images were converted to 8 bits, and the threshold was set to capture
the total structure and calculate the area of the spheroids.
employed for three-dimensional (3D) invasion assay, following the
manufacturer’s instructions. Spheroids of A375 transfected cells were
generated as previously reported by plating 500 cells for 48 h in 3D culture
qualified 96-well plate [36 (link)]. Then, spheroids were embedded into the Cultrex spheroid
invasion matrix. After 1 h at 37 °C, SFM with or without broad-spectrum
matrix metalloprotease inhibitor, Ilomastat/GM6001 (Cat# GM6001;
Millipore, Burlington, Massachusetts, USA) or uPAR inhibitor, M25 peptide, was
added to spheroids wells. Plates were incubated for 7 days, and spheroids were
photographed every 24 h through the use of Bio-Rad ZOE fluorescent cell imager
(Bio-Rad). ImageJ software (
Captured images were converted to 8 bits, and the threshold was set to capture
the total structure and calculate the area of the spheroids.
7
Transwell Migration and Wound Healing Assays
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For transwell migration assay, 5 × 104 cells were seeded in serum-free media into the upper chamber of 24 Well ThinCert Cell Culture Inserts (Greiner Bio-One S.r.l, Cassina de Pecchi, Italy). The lower well contained medium with 10% FBS. After 6 h incubation at 37 °C, cells remaining on the top side of the membrane were removed, and migrated cells stained, photographed, and counted. From each transwell, several images were acquired by using the light channel of Bio-Rad ZOE fluorescent cell imager (Bio-Rad Laboratories).
For wound healing assay, 1.8 × 103 melanoma or HFF cells were seeded in Culture-Insert 2 Well in µ-Dish 35 mm (ibidi GmbH, Gräfelfing, Germany). After 24 h, septa were removed and the images relative to starting point (T0) were acquired with Nikon Eclipse Ts100 phase-contrast microscope at ×4 magnification. The closure of the septa caused by the migrating melanoma cells was evaluated by acquiring images after 6 h and the distance between the two cell fronts was measured. As regarding HFF cells, after 24 h medium was replaced with CM derived from 24 h of melanoma cultured cells, septa were removed and the images were acquired to evaluate the closure of the septa. In all experiments, the CM used for stimulating HFF was normalized to the number of melanoma adherent cells.
For wound healing assay, 1.8 × 103 melanoma or HFF cells were seeded in Culture-Insert 2 Well in µ-Dish 35 mm (ibidi GmbH, Gräfelfing, Germany). After 24 h, septa were removed and the images relative to starting point (T0) were acquired with Nikon Eclipse Ts100 phase-contrast microscope at ×4 magnification. The closure of the septa caused by the migrating melanoma cells was evaluated by acquiring images after 6 h and the distance between the two cell fronts was measured. As regarding HFF cells, after 24 h medium was replaced with CM derived from 24 h of melanoma cultured cells, septa were removed and the images were acquired to evaluate the closure of the septa. In all experiments, the CM used for stimulating HFF was normalized to the number of melanoma adherent cells.
8
Cell Adhesion on Fibronectin and Mesothelial Cells
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Cells (15 × 103) after trypsinization were labeled with PKH67 green or PKH26 red fluorescent cell linker (Cat# MINI26-1KT; Sigma-Aldrich) for 5 min at 37 °C, washed twice, and added to a 96-well microplate, respectively, coated with fibronectin or a monolayer of human primary mesothelial cells on fibronectin. Cells were incubated with serum-free medium as indicated, and after 30 min, non-adherent cells were removed by washing three times with serum-free medium. Adherent cells were photographed using a Bio-Rad ZOE fluorescent cell imager (Bio-Rad Laboratories), and the results of the analysis of individual photos are reported. The number of adherent cells was quantified using the “Find Maxima” tool in FIJI software (noise tolerance = constant for the same set of images, output type = maxima within tolerance).
9
Imaging U251 Glioblastoma Cells
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A Bio-Rad ZOE fluorescent cell imager (Bio-Rad Laboratories, Hercules, CA, USA) was used for the bright field live images of human U251 glioblastoma cells.
10
Spheroid Live-Dead Assay Protocol
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Cells (1 × 103) were cultured in complete medium in a U-shaped bottom 96-well plate and centrifuged at 200 g for 5 min. 24 h after spheroid formation, serum-free medium, as indicated, was added and left for 72 h at 37 °C. After 72 h, the Cyto3DTM Live–Dead Assay Kit (TheWell Bioscience, Inc., North Brunswick, NJ, United States) was used to determine the live/dead nucleated cells using a dual-fluorescence system of acridine orange (AO) and propidium iodide (PI), both nuclear staining (nucleic acid binding) dyes. All live nucleated cells fluoresce green, and all dead nucleated cells fluoresce red. Several images were obtained using a Bio-Rad ZOE fluorescent cell imager under a phase-contrast microscope (Bio-Rad Laboratories). Image analysis was performed using the FIJI software by calculating the mean gray value of the green and red channels separately, and then the green/red ratio was calculated.
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