The live and dead assay was carried out to study cell proliferation and viability [38 (link)]. The samples were evaluated by using a live and dead cell imaging kit (Invitrogen, Carlsbad, CA, USA). The hydrogels were washed with PBS 3 times and were transferred to a confocal dish (Coverglass bottom dish, SPL Lifesciences, Pocheon-si, Korea). The hydrogels were stained with calcein AM and ethidium homodimer and incubated in the cell culture incubator (5% CO2 and 37 °C) for 30 min. The cell images were obtained using a super-resolution conformal laser scanning microscope (LSM 880, Airyscan, Carl Zeiss, Jena, Germany) along with the Z-stack process.
Cover glass bottom dish
Manufactured by SPL Life Sciences
Sourced in United States
The cover glass bottom dish is a laboratory equipment item designed to provide a transparent glass surface for cell culture and microscopy applications. It offers a stable and uniform platform for observing and analyzing cellular structures and behaviors under a microscope.
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Lab products found in correlation
Airyscan, by Zeiss (3 mentions)
Live dead cell imaging kit, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 568, by Abcam (2 mentions)
Igf1r, by Santa Cruz Biotechnology (2 mentions)
Alexa fluor 647, by Abcam (2 mentions)
Nedd4 1, by Santa Cruz Biotechnology (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Mitotracker red, by Thermo Fisher Scientific (1 mentions)
Mitotracker green, by Thermo Fisher Scientific (1 mentions)
Lsm710 confocal microscopy, by Zeiss (1 mentions)
Eclipse, by Nikon (1 mentions)
Er tracker red, by Thermo Fisher Scientific (1 mentions)
Fv1000 zcd laser scanning confocal system, by Olympus (1 mentions)
Fluoview version 2, by Olympus (1 mentions)
Mitosox, by Thermo Fisher Scientific (1 mentions)
Rhod 2 am, by Thermo Fisher Scientific (1 mentions)
Fluo 4 am, by Thermo Fisher Scientific (1 mentions)
S0033, by Beyotime (1 mentions)
13 protocols using cover glass bottom dish
1
Live/Dead Cell Proliferation Assay
2
Visualizing Mitochondrial Dynamics in Live Cells
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To visualize mitochondria, 125 nM of MitoTracker Red (Molecular Probes, Eugene, OR, USA) was added to cultured media and incubated cells for 30 min at 37 °C. After cells were washed with phosphate-buffered saline for three times, the mitochondria-stained cells were fixed with 4% paraformaldehyde solution for 10 min and rinsed with a mixture of phosphate-buffered saline: methanol (1 : 1). The fixed cells were permeabilized with methanol for 20 min at −20 °C. For immune fluorescence staining, cells were blocked with 1% bovine serum albumin in phosphate-buffered saline for 1 h at room temperature, followed by incubation with appropriate primary antibodies (anti-c-Myc antibody) overnight at 4 °C, and subsequently immunostained with an Alexa 488-conjugated secondary antibody. For mitochondria imaging in live cells, cells were seeded in a coverglass-bottom dish (SPL Life Sciences, Pochun, Korea) and mitochondria were stained with 100 nM of MitoTracker Green (Molecular Probes) for 30 min. Images were captured and analyzed using LSM 710 Confocal Microscopy (Carl Zeiss, Welwyn Garden City, UK). For time course analysis, after HeLa cells were transfected with YFP-mito and treatment of different concentrations of AMA, images were analyzed after 3 h of AMA treatment using fluorescence time-lapse microscope (Nikon eclipse, Nikon, Tokyo, Japan).
3
Bimane-α-MMC and ER Tracker Imaging
4
Calcium and ROS Imaging Assays
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The cells were seeded in a cover glass‐bottom dish (SPL Life Sciences Co., Ltd., Korea). Calcium mobilization analysis was performed using Fluo‐4 AM (acetoxymethyl) ester (Invitrogen, San Diego, CA, USA) or Rhod‐2 AM (Invitrogen, San Diego, CA, USA), as described in the recent report.[12 ] Intracellular ROS were measured by flow cytometry using a cell‐based ROS assay kit (S0033; Beyotime Biotechnology, Beijing, China) and MitoSOX (M36008, Invitrogen, USA). Details of the procedure are available in Appendix A1 in the Supporting Information.
5
Real-time Imaging of CAR-T Cell Dynamics
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5 × 104 293-ACE2-mCherry cells were resuspended with 500 μl RPMI-1640 medium (5% FBS, phenol red free) and pre-plated in a cover-glass bottom dish (SPL life sciences) at 37°C. After being cultured overnight, 1 μg/ml RBD protein was added to cells and incubated at 37°C for 1 h. Then 2×106 CR3022-8a-28Z-EGFP CAR-T cells were resuspended with 1.5 ml RPMI-1640 medium (5% FBS, phenol red free) and added to the dish for co-culture in an inverted fluorescence microscope with climate control (Zeiss Observer Z1). Images were captured from the beginning of the co-culture with a 2 min interval for 24 h. Videos were exported and analysed by using ZEN Lite software (Zeiss).
6
Spheroid Viability and Morphology Analysis
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Viability,
cytotoxicity, and morphology were analyzed under a super-resolution
confocal laser scanning microscope (LSM 880 with Airyscan, Carl Zeiss,
Germany) in Z-stack mode and a Bio-LV SEM. A live/dead cell imaging
kit (Invitrogen, USA) was applied following the manufacturer’s
instruction. The spheroids were cultured for 7 days, transferred to
a confocal dish (cover glass bottom dish, SPL, South Korea), and treated
with the staining reagent. The spheroids were incubated under standard
cell culture conditions (37 °C and 5% CO2) for 30
min, and the images were observed. Live cells and dead cells were
stained with calcein-AM (green) and an ethidium homodimer (red), respectively.
Furthermore, for the SEM observation, the spheroids were cultured
for 7 days and were fixed with 2.5% glutaraldehyde (Sigma-Aldrich,
USA) for 24 h at 4 °C. The cross-sectioned spheroids were gold-sputtered
under a vacuum condition.
cytotoxicity, and morphology were analyzed under a super-resolution
confocal laser scanning microscope (LSM 880 with Airyscan, Carl Zeiss,
Germany) in Z-stack mode and a Bio-LV SEM. A live/dead cell imaging
kit (Invitrogen, USA) was applied following the manufacturer’s
instruction. The spheroids were cultured for 7 days, transferred to
a confocal dish (cover glass bottom dish, SPL, South Korea), and treated
with the staining reagent. The spheroids were incubated under standard
cell culture conditions (37 °C and 5% CO2) for 30
min, and the images were observed. Live cells and dead cells were
stained with calcein-AM (green) and an ethidium homodimer (red), respectively.
Furthermore, for the SEM observation, the spheroids were cultured
for 7 days and were fixed with 2.5% glutaraldehyde (Sigma-Aldrich,
USA) for 24 h at 4 °C. The cross-sectioned spheroids were gold-sputtered
under a vacuum condition.
7
Cellular ROS Quantification using Carboxy-H2DCFDA
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Cellular ROS levels were examined using a cell-permeable fluorogenic probe, carboxy-H2DCFDA. The cells were seeded in a cover glass bottom dish (SPL Life science) and further incubated for overnight. After the Pba-laser therapy, cells were washed with PBS and incubated with 30 µM of carboxy-H2DCFDA for 30 min at 37°C. For the nuclei staining, H342 was incubated for 10 min. Then confocal images were obtained and green fluorescent intensities were quantified using a LSM 710 confocal microscope and ZEN2011 software.
8
Antifungal Effect on Candida albicans Biofilm
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C. albicans mid-log phase cultures (3 mL/dish, 6 × 106 cells/mL) were transferred to a cover glass bottom dish (SPL lifescience, Pocheon, Korea) and incubated for 48 hours before being treated with either HBD3-C15 (0, 12.5, 25, 50, 100, 150, 200, and 300 µg/mL), aqueous CH (100 µg/mL) or Nys (20 µg/mL) triplicate dishes of each for 7 days at 37℃. Each dish was aspirated to remove broth, and washed gently with PBS. Finally they were stained with the FilmTracer LIVE/DEAD Biofilm viability kit (Molecular Probes, Carlsbad, CA, USA), which uses SYTO9 and propidium iodide (PI) to stain live and dead cells within biofilms respectively. SYTO9 stains both live and dead microorganisms fluorescent-green, whereas PI stains only the nucleic acids of cells with damaged membranes, and thereby identifies dead microbes. The stained C. albicans biofilms were examined by confocal laser scanning microscopy (CLSM, LSM 700, Carl Zeiss, Jena, Germany) with the ×40 lens. CLSM images were acquired by using ZEN 2010 (Carl Zeiss) software at a resolution of 512 × 512 pixels with a zoom factor of 2.0. Each 2 dimensional (2D) image covered an area of 230.34 × 230.34 µm. The 3 dimensional (3D) reconstructed images had a z step of 1 µm in each stack, and there were 15 stacks in total.
9
Live/Dead Assay of hBMSC-Encapsulated Hydrogels
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A live/dead assay was conducted to evaluate the cell viability and cytotoxicity. The hBMSC-encapsulated samples were incubated under standard culture conditions (5% CO2 and 37 °C) for 7 days. A live/dead cell-imaging kit (Invitrogen, CA, USA) was used, following the kit protocol. Briefly, the degree of the middle height of the cylindrical hydrogel was cut, and then the cross-sections were transferred to a confocal dish (Cover glass-bottom dish, SPL Life Science, Pocheon, Republic of Korea) and stained with calcein-AM and ethidium homodimer and incubated in the cell culture incubator. The images were obtained by using a Super-Resolution Confocal Laser Scanning Microscope (LSM 880 with Airyscan, Carl Zeiss, Germany) with the process of Z-stack. All measured thicknesses were unified to 200 μm and the fluorescence intensity of live/dead staining was characterized by Image J software V.1.8.0.
10
CLSM Analysis of FITC-LDH-CO3 Uptake in MG63 Cells
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For CLSM measurements, MG63 cells in 2 mL of DMEM medium (2.0 × 106 cells per well) were seeded in Coverglass bottom dish (SPL Lifesciences Co., Ltd., Pocheon, Korea) and incubated at 37°C and 5% CO2 for 24 hours. Then, the medium in the well was replaced by the FITC-LDH-CO3 suspension (20 µg/mL) in fresh medium and incubated under the same condition. A control experiment was also performed with intact FITC (20 µg/mL) and the centrifugal supernatant of the FITC-LDH-CO3 suspended in the cell medium under the same conditions. After incubation for 1 hour, the cells were washed three times with DPBS and fresh cell medium were then added to the plates. For the staining of nucleus, 140 µL of NucBlue (Live Cell Stain ReadyProbes® reagent; Thermo Fisher Scientific, Waltham, MA, USA) was added and incubated for additional 20 minutes at 25°C. The stained cells were imaged under a confocal laser scanning microscope (Leica TCS SP8 STED CW; Leica Microsystems Inc., Buffalo Grove, IL, USA) equipped with both DAPI filter (Ex. 420 nm/Em. 460 nm) and FITC filter (Ex. 495 nm/Em. 520 nm). The obtained images were processed using Leica LASAF software.
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