Product Introduction
hNTCP-expressing Huh7D(Huh7D-hNTCP), based on hepatocellular cancer cell lines can be personalized to customize the cell lines.
Catalog:T25 flask or cryopreservation tube
Quotation:Please contact on 19902901483
(Can be attachment,Cat# LV-NTCP002)
(For research use only)
I Introduction
hNTCP-expressing Huh7D (Huh7D-hNTCP) was developed by a laboratory of the Chinese Academy of Sciences and authorized by our company for cell sharing. Huh7D is an excellent cell line selected from several strains of Huh7 cells by 2.5% DMSO and 4% PEG8000 tolerance experiments, the main advantage is that under the induction of 2.5% DMSO, the cells are dense monolayer, cell polarization and cell differentiation, long-term maintenance, tolerance to 4% PEG8000 toxicity, etc. By lentivirus-mediated overexpression of the hepatitis B virus receptor hNTCP, cells are well preserved in cellular traits. High expression of hNTCP has excellent susceptibility to hepatitis B virus. This cell is for research use only and can’ t be transferred to the third parties for use unless authorized, or peril.
II Reagents and Materials
-hNTCP-expressing Huh7D(Huh7D-hNTCP)
-Conventional medium (DMEM +10% FBS+1% PS)
-Screening medium (conventional medium +5ug/mL Puromycin)
-Sustaining medium (DMEM +10% FBS+1%PS+2.5% DMSO)
-PBS
-0.5% pancreatin
-Culture flask
-Pipette tip and pipette
-Thermostat water bath (preheating at 38°C)
-Biosafety cabinet
-Centrifuge
-37°C/5% CO2 Incubator
-75% Alcohol
III Cell Resuscitation and Plating
1. Sterilize the Biosafety cabinet with UV for 15 minutes, and preheat the medium in a 38 °C thermostat water bath.
2. The frozen cells should be transferred quickly from the refrigerated position to thermostatic water bath of 38 ° C . Immerse them as much as possible in 38 ° C water and shake it horizontally. But make sure that the cap of the freezing tube is kept above the surface of the water.
3. Defrost the freezing tube for about 90-120 seconds until only a small amount of crushed ice floats in the tube.
4. Sterilize the freezing tube with 75% alcohol and transfer it to a biosafety cabinet.
5. Centrifuge at 300 × g for 5 min at 4 °C. Remove the supernatant and resuspend with regular medium.
6. According to experience, the pelleted cells can be resuspended with conventional medium and be fixed the volume of 4 ml. The survival rate and total cell volume of cells can be measured by trypan blue exclusion.
7. Cells are seeded into culture flasks at a density of 2×104 cells/cm2. Shake well and incubate in a 37 °C/5% CO2 incubator. Change the solution after 24 hours, and then every other day.
IV Cell Culture, Passage and Screening
1. The cells can be passaged when the confluency reaches 80%.
2. Aspirate the old culture solution and add a small amount of PBS to wash the cells. In addition, add an appropriate amount of pancreatin so that the amount of pancreatic enzyme can cover the cells. Incubate the cells at 37°C for 2-3 min. Then, terminate digestion by conventional medium.
3. Collect the cell suspension and centrifuge at 300 × g for 5 min at 4 °C. Remove the supernatant and resuspend with conventional medium.
4. Cells can be passaged at a ratio of 1:3 or 1:4. The cell suspension should be seeded into a new cell flask and cultured in a 37 °C/5% CO2 incubator. The growth of the cell adhesion can be observed every other day.
5. In order to achieve better infection efficiency, the cell line needs to be screened. After screening for 1 week, the selected cells could be preserved for subsequent experiments.
Please refer to the article for hepatitis B virus infection
PMID: 28971350,DOI: 10.1007/s12250-017-3983-x
V Customer Service
If you find any quality problems with the product, please collect the original data and contact the company's salesmen or technical support at the first time. The company ensure after-sales service. Every laboratory has different conditions, different operating habits, different proficiency, and objective factors in experimental failure. If the operation is not strictly in accordance with the instructions or exceeds the time limit of after-sales, the company does not do after-sales. Please understand and support us.
Validity period and raw data provided:
Resuscitation problems: Within 24 hours of resuscitation, trypan blue staining or PI staining should be provided.
Pollution problems: Within 96 hours of resuscitation, microscope photographs of differences should be provided.
Purity issues: Within one month, immunofluorescence or flow cytometry results should be provided
VI Contact Number
Tel:0755-28284050
Technical Support:19902901483 (Dr. Zhou)