HeLa cells (7.0 × 104 per dish) were seeded on 20 mm confocal imaging dishes (glass bottom dish, NEST) and stabilized for 72 h. HeLa cells were incubated with 1 μM ACC1 and ACC2 for 30 min and a two-photon PDT experiment was performed. After PDT treatment, the HeLa cells were incubated with 10 μM propidium iodide (PI) for 30 min. Afterward, cells were washed and the images were taken using a confocal microscope with excitation at 552 nm and emissions of 650–700 nm (for PI). To calculate the dead cell ratio more accurately, 1 μM Hoechst 33342 was stained with PI, and the corresponding ratio (live/dead) was calculated using the number of blue and red fluorescent dots.
Glass bottom dish
Manufactured by NEST Biotechnology
Sourced in China
The Glass Bottom Dish is a laboratory equipment designed for cell culture applications. It features a transparent glass bottom that allows for high-quality imaging and observation of cells under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
Sylgard 184 kit, by Dow (2 mentions)
Hematoxylin, by ZSGB-BIO (1 mentions)
Leica tcs sp5, by Leica camera (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Matrigel, by Corning (1 mentions)
Volocity software, by PerkinElmer (1 mentions)
Ultraview, by PerkinElmer (1 mentions)
Ptflc3 plasmid, by Addgene (1 mentions)
Lsm 780, by Zeiss (1 mentions)
Chambered coverglass, by Thermo Fisher Scientific (1 mentions)
Tcs sp8 microscope, by Leica camera (1 mentions)
Ab5408, by Abcam (1 mentions)
Ab5095, by Merck Group (1 mentions)
Anti γ tubulin, by Merck Group (1 mentions)
Airyscan mode, by Zeiss (1 mentions)
10 protocols using glass bottom dish
1
Two-Photon PDT Cytotoxicity Assay
2
Quantifying Abrin Internalization with S008
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A 2×105 Vero cells in 1 mL complete DMEM were allowed to adhere to a glass bottom dish (NEST, Cat. No.:801002) at 37°C overnight and then washed twice with pre-cooled PBS at 4°C. A 1×106 Jurkat cells were harvested and washed twice with pre-cooled PBS. All cells were blocked with TruStain FcXTM (anti-mouse CD16/32 and anti-Human IgG receptors) for 10 min at RT and then incubated with Alexa-488 labeled Abrin (0.1 mg/mL) in the presence or absence of Alexa-594 labeled S008 (molar ratio of abrin: S008 as 1:1, 1:10, 1:100) or with Alexa-594 labeled S008 alone for 1 h at 37°C. Cells alone were set as blank controls. Samples were washed twice with pre-cooled PBS, fixed with pre-cooled 4% paraformaldehyde fixative followed by being washed with pre-cooled PBS once. Nuclei were stained with antifade mounting medium containing DAPI for at least 2 min. Cells were visualized and images were acquired using a Zeiss LSM 880 Meta Laser Confocal scanning microscope (Carl Zeiss Foundation, Germany) and analyzed using the Zeiss LSM image browser to visualize the internalized antibody.
3
Immunofluorescence and Immunohistochemistry of LSR
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For immunofluorescence in Figure 3 Supplementary Table 1 Supplementary Table 1
4
Actin Dynamics in Solid Matrigel
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Three to five celibate young males were dissected in 2 μl culture medium containing 2 μM SiR-actin on a glass-bottom dish (NEST 801002). A piece of wet tissue was placed in the dish to prevent medium evaporation. The dish was kept in the dark for 5 min at 20°C before adding 2 μl Matrigel (Corning 356234) to make the medium solid. Images in 13 z series (0.5 μm/section) were captured every 30 s for 30 min at 20°C using a spinning-disk confocal scanner unit (UltraView; PerkinElmer). The amount of 10 μl 5% NP-40 (Fluka 74358) was added on top of the solid medium after the first time point. Images were viewed and analyzed using Volocity software (PerkinElmer).
5
Autophagy Assessment in Pancreatic Cancer Cells
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To perform this assay, 1 × 105 6606PDA cells per well or 4 × 105 MIA PaCa-2 cells per well were plated in a glass-bottom dish (NEST, Wuxi, China, code 801001). On the following day, the cells were transfected with the ptfLC3 plasmid (Addgene, Cambridge, UK, code 21074), which was a kind gift from Tamotsu Yoshimori [17] (link), and Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, USA, code L3000001). After 24 h, the cells were treated with medium containing DMSO (Sham) or LW6 (80 µM for MIA PaCa-2, 160 µM for 6606PDA) for another 12 h and then were fixed with 4% formalin. The nuclei were stained with 4′6-diamidino-2-phenylindole (DAPI). The images were acquired by a confocal microscope, Zeiss LSM 780 (Zeiss, Oberkochen, Germany), using the 60× oil objective. Only cells that were transfected with the ptfLC3 plasmid were evaluated. To evaluate the ratio of autophagosomes/cell in each field, the following formula was used: autophagosomes/cell = the number of yellow dots/the number of nuclei. Similarly, the ratio of autolysosomes/cell was defined as the number of red dots divided by the number of nuclei in each field. For each experiment, five fields per treatment were randomly acquired and evaluated.
6
Generating 3D Multicellular Tumor Spheroids
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Three-dimensional (3D) MCTSs were prepared using the liquid overlay method. A trypsin/EDTA solution was used to separate HeLa cells in the exponential growth stage into single-cell suspensions. HeLa cells (2 × 105) were transferred to 1.5% agarose-coated transparent 25-well plates with 30 μL of Dulbecco's modified Eagle medium (DMEM) containing 10% serum. The single HeLa cells in the wells generated approximately 2400 μm in diameter of MCTSs on day 4 with 5% CO2 in air at 37 °C in an incubator chamber. After generation of HeLa MCTSs, each MCTS in a 25-well plate was transferred to 20 mm confocal imaging dishes (glass bottom dish, NEST) and imaged using a 40× objective with xyz mode to monitor the volume of the MCTS.
7
PDMS Mold and Cell Network Culture
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The preparation processes of PDMS mold and cell network culture have been introduced in our recent work (Ouyang et al., 2020 (link)). Briefly, a thin layer of PDMS (∼600 μm in thickness) was generated by mixture of the two liquid components (10:1) from the Sylgard 184 kit (Dow Corning). The PDMS sheet was cut into circular pieces, on which one or more holes with 0.6 cm in diameter were created by a mechanical puncher. The PDMS mold was sterilized and attached onto the glass-bottom dish (NEST). Matrigel containing 0.5 mg/ml COL was added into the PDMS mold on ice, and then placed into the incubator to solidify at 37°C for 30 min. To seed cells, about 30 μL of cell suspension was added on top of the hydrogel and stayed for 30 min in the incubator before the addition of more culture medium.
8
PDMS Mold for 3D Cell Culture
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The PDMS mold and cultivation patterns of the cell mesh structure were described in our previous study [22 (link)]. A thin layer of PDMS (~500 μm in thickness) was generated as follows. First, the two liquid components included in the Sylgard 184 kit (Dow Corning, Midrand, MI, USA) were mixed thoroughly at a mass ratio of 10:1. Then, this mixture was added to the tablet, air bubbles were removed under vacuum, and the sheet was cured in an oven at 70 °C for 3 h. The PDMS sheet was cut into pieces of appropriate size, and a hole 0.6 cm in diameter was created in the middle using a mechanical puncher. Then, the PDMS was sterilized overnight under a UV lamp in a cell culture hood and attached to the center of a glass-bottom dish (NEST). The liquid mixture was prepared using 100% Matrigel (~10 mg/mL) and pH-neutralized 1 mg/mL COL at a volume ratio of 1:1 on ice. Then, ~50% Matrigel containing 0.5 mg/mL COL was added to the PDMS mold on ice. The assembly was placed into an incubator and allowed to solidify at 37 °C for 30 min. Then, 10–20 μL of cell suspension was added to the top of the hydrogel, and the mold was placed into the incubator for 30 min, followed by the addition of additional culture medium.
9
FRAP Analysis of LLPS and SGs
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All FRAP experiments were performed on a Leica TCS SP8 microscope with 100× oil objective by using the FRAP module. For protein samples, 20 μL LLPS sample of Hsp70 or its domains was placed in a glass bottom dish (Nest, 80100). FRAP images were acquired at 488 nm and performed with 1,024 × 1,024 pixel. Bleaching area of droplet was restricted to a graphical region and then quenched for 2.58 s at full laser power. After photo bleaching, images were obtained at 2.58 s/frame.
For living cells assay, HeLa cells were grown in chambered coverglass (Thermo Fisher Scientific, 155,383), and then transfected with mEGFP-G3BP1 or RFP-FUS R521H. Cells were treated with 100 μM of sodium arsenite for indicated time to induce SGs after culturing for 24 h. SGs were bleached by a 488 nm or 561 nm laser at 100% power and then acquired time-lapse images at 2.58 s per frame for indicated time. Data analysis were processed by Fiji and GraphPad Prism. All experiments were repeated independently for more than three times. All statistic values were displayed as mean ± S.D. The statistical significance in this study is determined by the unpaired, two-tailed Student’s t test. p > 0.05 is set to not significant; ∗p < 0.05; ∗∗p < 0.01.
For living cells assay, HeLa cells were grown in chambered coverglass (Thermo Fisher Scientific, 155,383), and then transfected with mEGFP-G3BP1 or RFP-FUS R521H. Cells were treated with 100 μM of sodium arsenite for indicated time to induce SGs after culturing for 24 h. SGs were bleached by a 488 nm or 561 nm laser at 100% power and then acquired time-lapse images at 2.58 s per frame for indicated time. Data analysis were processed by Fiji and GraphPad Prism. All experiments were repeated independently for more than three times. All statistic values were displayed as mean ± S.D. The statistical significance in this study is determined by the unpaired, two-tailed Student’s t test. p > 0.05 is set to not significant; ∗p < 0.05; ∗∗p < 0.01.
10
Immunofluorescence and Live-Cell Imaging
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Cells were quickly rinsed with pre-warmed DPBS, xed with pre-cooling methanol for 10 min at -20°C, and permeabilized in 0.5% Triton X-100 for 10 min. Cells were then blocked with 5% Bovine Serum Albumin for 30 min and incubated overnight at 4°C with anti-RNAPII phospho S5 (1:1500, Abcam, ab5408), anti-RNAPII phospho S2 (1:3000, Abcam, ab5095), anti-γ-Tubulin (1:1000, Sigma, T6557 For live imaging, cells in glass bottom dish (NEST, 801001) were transfected with pEGFP-Tubulin and mCherry-H2B using Lipofectamine 3000 (Invitrogen), and synchronized with double thymidine block procedure. Cells were then released into thymidine-free medium for 8 hours and imaged in PeCon environmental microscope incubator (ZEISS) at 37°C and 5% CO 2 . Images were collected on the LSM 880 confocal microscope using a 63× oil immersion objective lens with Airyscan mode (ZEISS).
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