Huh7 cells were cultured on a 20-mm glass bottom dish (NEST) and transfected with the indicated plasmids. Then the cells were washed three times with PBS, fixed with 4% paraformaldehyde–containing PBS for 20 min, and permeabilized with 1% Triton X-100–containing PBS for 20 min. After being blocked with 2% bovine serum albumin–PBS for 1 h, the cells were incubated with primary antibody diluted in antibody dilution buffer overnight at 4°C.After being washed three times with PBS, the cells were incubated with second antibody for 1 h and washed with PBS for three times. Then the cells were treated with DAPI (Beyotime) for 10 min, washed three times with PBS, and observed with a confocal microscope (Olympus). The following primary and second antibodies were used: mouse anti-NS5A antibody 9E10 was kindly provided by Chaoyang Li (Wuhan Institute of Virology, Chinese Academy of Sciences, China); mouse anti-Flag antibody was purchased from Beyotime; rabbit anti-Myc antibody was purchased from Proteintech; TRITC-conjugated or FITC-conjugated second antibodies were purchased from Pierce.
20 mm glass bottom dish
Manufactured by NEST Biotechnology
Sourced in China
The 20-mm glass bottom dish is a laboratory instrument designed for various microscopy applications. It features a glass bottom that allows for high-quality optical imaging and observation of samples.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Lsm900, by Leica (2 mentions)
Mouse anti flag antibody, by Beyotime (1 mentions)
Rabbit anti myc antibody, by Proteintech (1 mentions)
Click it edu imaging kit, by Thermo Fisher Scientific (1 mentions)
Laser confocal fluorescence microscopy, by Leica (1 mentions)
Ros id hypoxia oxidative stress detection kit, by Enzo Life Sciences (1 mentions)
Lsm 710 confocal laser scanning microscope, by Zeiss (1 mentions)
6 protocols using 20 mm glass bottom dish
1
Immunofluorescence Assay for HCV Proteins
2
Evaluating BMSC Proliferation Dynamics
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To study the proliferative ability of the BMSCs, cell growth was evaluated by manually counting at each passage. Briefly, 3 × 105 BMSCs were seeded in the 60 mm2 dish and passaged once the culture reached 80–90% confluence. Cell numbers were recorded and cells were replated at the same density each time. Cell proliferation was also examined by EdU assay. In brief, 1.5 × 105 BMSCs were plated in 20 mm glass bottom dish (NEST Biotechnology, China) and cultured overnight. After incubation with EdU for another 36 h, the cells were fixed in 4% paraformaldehyde for 15 min at room temperature, washed twice with 3% BSA in PBS, incubated with 0.5% Triton X-100 in PBS for 20 min and stained with the Click-iT EdU Imaging Kit (Life Technologies) according to the manufacturer’s instruction. Nuclear staining was performed with 4′, 6-diamidino-2-phenylindole (DAPI). Photomicrographs were taken by Laser confocal fluorescence microscopy (Leica, Germany). The percentage of proliferating cells was calculated based on counting EdU-positive cells/total cells in five individual visual fields.
3
Macrophage Endosomal Acidification and PW Uptake
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THP-1 cells (2.4×105 cells/well) were seeded in a 20 mm glass bottom dish (NEST, Wuxi, China) and differentiated into macrophages. To assess the endosomal acidification, cells were incubated with pHrodo red-labeled dextran (10,000 MW, 10 µg/mL) and PW (200, 100 and 50 nM) or chloroquine (30 µM) for 5 h and processed for confocal imaging. To examine the uptake of PW in macrophages, cells were incubated with Cy5-labelled PW (PW-Cy5, 10 nM) for 5 h, and the cell membrane and nucleus were then stained with DiO and DAPI, respectively. Cells were washed with PBS and imaged on a confocal microscope (LSM900, Leica Microsystems Inc., Wetzlar, Hessen, Germany). The fluorescence of pHrodo red (ex: 565 nm; em: 585 nm) and Cy5 (ex: 640 nm; em: 670 nm) in the cells was quantified by the ImageJ software. For each condition, at least 30 cells were quantified with three independent repeats.
4
Cellular Uptake and Acidification Dynamics
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THP‐1 cells (2 × 105 cells) were seeded in a 20‐mm glass bottom dish (NEST, Wuxi, China) and differentiated into macrophages. They were incubated with pHrodo red‐ and fluorescein‐labeled 10000 MW dextran (10 µg mL−1) (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h before OM (500 µg mL−1) or chloroquine (30 µM) treatment for 3.5 h. Cells were then washed 3 times with PBS and imaged on a confocal microscope (LSM900, Leica, Wetzlar, Hessen, Germany). The fluorescence intensity of pHrodo red (ex: 565 nm; em: 585 nm) and fluorescein (ex: 488 nm; em: 525 nm) in the cells was quantified by Image J software (NIH, Bethesda, MD, USA). For each condition, at least 30 cells from 3 independent experiments were quantified to obtain the intensity ratio of fluorescein to pHrodo red.
To examine the cellular uptake of Nano‐OM, THP‐1 cell‐derived macrophages were treated with DiD (Solarbio, Beijing, China)‐labeled liposomes for localizing Nano‐OM in the cells, and stained with 25 µM DiO (Beyotime, Shanghai, China) for 20 min for labeling the cell membranes and DAPI (Beyotime, Shanghai, China) for 5 min for labeling the nucleus; the fluorescence images were acquired on a confocal microscope (LSM900, Leica, Wetzlar, Hessen, Germany).
To examine the cellular uptake of Nano‐OM, THP‐1 cell‐derived macrophages were treated with DiD (Solarbio, Beijing, China)‐labeled liposomes for localizing Nano‐OM in the cells, and stained with 25 µM DiO (Beyotime, Shanghai, China) for 20 min for labeling the cell membranes and DAPI (Beyotime, Shanghai, China) for 5 min for labeling the nucleus; the fluorescence images were acquired on a confocal microscope (LSM900, Leica, Wetzlar, Hessen, Germany).
5
Nanoparticle Cellular Uptake and ROS
6
Cellular Uptake and Acidification Assessment
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THP‐1 cells (2.4 × 105 cells) were seeded in a 20 mm glass bottom dish (NEST, Wuxi, China) and differentiated into macrophages as previously described. To examine the cellular uptake of M‐P12, macrophages were treated with DiD‐labelled M‐P12 (0.2 mg mL−1 phospholipid and 0.77 µg mL−1 DiD) overnight, and stained with DiO for 20 min and DAPI for 5 min to label the cell membrane and nucleus, respectively. The fluorescence images were acquired on a confocal microscope (LSM900, Leica, Wetzlar, Hessen, Germany).
To assess the endosomal acidification, macrophages were incubated with pHrodo red (10 µg mL−1) and fluorescein‐labelled dextran (20 µg mL−1) in the presence of nanomicelles (phospholipid: 0.2 mg mL−1) or chloroquine (CHQ, 30 µm ) treatment for 6 h. Cells were then washed 3 times with PBS and imaged on a confocal microscope (LSM900, Leica, Wetzlar, Hessen, Germany). The fluorescence intensities of pHrodo red (ex: 565 nm; em: 585 nm) and fluorescein (ex: 488 nm; em: 525 nm) were quantified by Image J software. For each condition, at least 60 cells from 3 independent experiments were quantified to obtain the intensity ratio of fluorescein to pHrodo red.
To assess the endosomal acidification, macrophages were incubated with pHrodo red (10 µg mL−1) and fluorescein‐labelled dextran (20 µg mL−1) in the presence of nanomicelles (phospholipid: 0.2 mg mL−1) or chloroquine (CHQ, 30 µ
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