To demonstrate the synergistic hypoxia relief ability of AIO, AIP, and Oxygen tank, an oxygen probe (OX-NP, 1.6 × 40 mm-needle sensor for piercing, Unisense A/S CO.LTD) was used to measure the oxygen concentration of culture media of different groups. To further measure the hypoxia reverse ability of the Oxygen tank, AGS cells were seeded into confocal wells (20mm-glass -bottom dishes, NEST Biotechnology Co. Ltd.) at a density of 2.5 × 104 cells per well. After 24 h incubation, corresponding wells were placed in the hypoxia incubator (10% O2) to simulate a hypoxia condition. After being washed 3 times, the cells were evaluated by hypoxia-inducible factor-1α.
20 mm glass bottom dishes
Manufactured by NEST Biotechnology
Sourced in China
20-mm glass-bottom dishes are laboratory equipment used for various cell culture and microscopy applications. They feature a glass bottom that allows for direct observation and imaging of cells under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Cell culture dishes plates, by NEST Biotechnology (2 mentions)
Hoechst 33342, by Solarbio (2 mentions)
Ox np, by Unisense (1 mentions)
Dithiothreitol (dtt), by Solarbio (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
Round coverslips, by NEST Biotechnology (1 mentions)
Anti caspase3 antibody, by ABclonal (1 mentions)
Anti β tubulin antibody, by ABclonal (1 mentions)
Anti cd31 antibody, by ABclonal (1 mentions)
Centrifuge tubes, by NEST Biotechnology (1 mentions)
Cck 8, by Meilun (1 mentions)
Sepharose cl 4b, by Solarbio (1 mentions)
Coomassie brilliant blue fast staining solution, by Solarbio (1 mentions)
Facscalibur, by BD (1 mentions)
Fv1200, by Olympus (1 mentions)
Confocal laser scanning microscope, by Olympus (1 mentions)
5 protocols using 20 mm glass bottom dishes
1
Synergistic Hypoxia Relief Ability
2
Nanoparticle-based Chemotherapeutic Delivery
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CPT, bis(2-hydroxyethyl) disulfide, 1,6-hexanediol, triphosgene, and 4-dimethylaminopyridine (DMAP) were purchased from Aladdin Co. Ltd. (Shanghai, China). Egg yolk lecithin and DSPE-PEG2k were obtained from Shanghai Advanced Vehicle Technology Co. Ltd. (Shanghai, China). Cell culture dishes/plates, round coverslips, 20-mm glass-bottom dishes, and centrifuge tubes were obtained from NEST Biotechnology Co. Ltd. (Wuxi, China). The total GSH assay kit was purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). DTT, Hoechst 33342, 4′,6-diamidino-2-phenylindole (DAPI), and TUNEL apoptosis assay kits were purchased from Beijing Solarbio Science & Technology Co. Ltd. (Beijing, China). The anti-Ki67 antibody was obtained from Biosynthesis Biotechnology Inc. (Beijing, China). The anti-CD44 antibody, anti-CD326 antibody, anti–caspase3 antibody, anti–β-tubulin antibody, and anti-CD31 antibody were purchased from ABclonal Biotechnology Co. Ltd. (Wuhan, China). CCK-8, DiR, and Bouin’s fluid were purchased from Dalian Meilun Biotechnology Co. Ltd. (Dalian, China). All other reagents used were of analytical grade.
3
Synthesis and Characterization of Pyropheophorbide a Nanoparticles
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Pyropheophorbide a (PPa) was purchased from Shanghai Xianhui Pharmaceutical Co., Ltd. (Shanghai, China). N,N-Dimethylglycine (DMG), N-methylmorpholine (NMM), 2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) were purchased from J&K Scientific Co., Ltd. (Beijing, China). 1, 2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-mPEG2k), cholesterol were obtained from Shanghai Advanced Vehicle Technology Co., Ltd. (Shanghai, China). Sepharose CL-4B, Coomassie Brilliant Blue Fast Staining solution and Hoechst 33342 were obtained from Solarbio Science & Technology Co., Ltd. (Beijing, China). The anti-P-selectin antibody (A1425), anti-CD61 antibody (A2542), anti-CD47 antibody (A1838), anti-CD31 antibody (A0378) and anti-Ki-67 antibody (A2094) were purchased from ABclonal Biotechnology Co., Ltd. (Wuhan, China). The anti-GPIbα antibody (bs-2347R) was obtained from Biosynthesis Biotechnology Inc. (Beijing, China). The anti-CD41 antibody (DF7456) and anti-HIF-1α antibody (AF1009) were purchased from Affinity Biosciences Ltd. (Cincinnati, OH, USA). Cell culture dishes/plates and 20 mm glass-bottom dishes were obtained from NEST Biotechnology Co., Ltd. (Wuxi, China).
4
Visualizing siRNA Nanoparticle Uptake
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SMMC-7721 cells were cultured with 1 mL DMEM containing 10% FBS on 20-mm glass-bottom dishes (NEST) at 5 × 104 cells/dish. After 24 h, the medium was exchanged with fresh medium, and NP/Cy3-siRNA, CaP/Cy3-siRNA and Cy3-siRNA were added to the dish (100 nM Cy3-siRNA) for different amounts of time. The SMMC-7721 cells were washed 3 times with PBS and were stained with Hoechst33342 for 5 min. The intracellular distribution of nanoparticles was visualized with a FV-1200 Olympus confocal microscope.
SMMC-7721 cells (1 × 105) were seeded onto 6-well plates with DMEM containing 10% FBS. After 24 h, the medium was replaced with fresh medium containing NP/FAM-siRNA nanoparticles (100 nM FAM-siRNA). At different time points, the cells were washed with PBS and detached with trypsin. The cellular uptake of FAM-siRNA was monitored using flow cytometry (FACSCalibur, BD Bioscience).
SMMC-7721 cells (1 × 105) were seeded onto 6-well plates with DMEM containing 10% FBS. After 24 h, the medium was replaced with fresh medium containing NP/FAM-siRNA nanoparticles (100 nM FAM-siRNA). At different time points, the cells were washed with PBS and detached with trypsin. The cellular uptake of FAM-siRNA was monitored using flow cytometry (FACSCalibur, BD Bioscience).
5
Cellular Uptake of DA-PLGA-PEG-cRGD Nanoparticles
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A laser confocal scanning microscope was used to evaluate the cellular uptake of DA in the HUVECs. Briefly, 1.0 × 104 HUVECs were cultured with 1 mL DMEM containing 10% FBS on 20-mm glass-bottom dishes (NEST) and incubated for 24 h at 37 °C with 5% CO2. After incubation, the HUVECs were treated with FITC + ox-LDL, FITC@DA-PLGA-PEG-cRGD NPs + ox-LDL, FITC@DA-PLGA-PEG-cRGD NPs + ox-LDL + US containing 20 µg/mL of FITC and 100 µg/mL ox-LDL for different times and were then rinsed three times with PBS. The cells were stained with Lyso-Tracker Red for 30 min at 37 °C with 5% CO2 and were washed 3 times with PBS. Subsequently, HUVECs were stained with Hoechst 33342 for 5 min and were washed 3 times with PBS again. Finally, the cells were fixed with 4% paraformaldehyde solution for 30 min at room temperature and were then examined using a confocal laser scanning microscope from the Olympus Optical Company, Ltd. (Tokyo, Japan) to determine cellular uptake of DA-PLGA-PEG-cRGD NPs.
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