Product Introduction
Primary Duck Hepatocytes
(Plateable, Cat# LV-PDH001)
(For research use only)
In order to ensure the safety and biosafety of the experimenters, please wear necessary protective equipment such as protective masks, latex gloves and protective eye shields (during revival) when contacting this product and its waste. Please operate in strict accordance with this manual, and the waste generated after the experiment must be harmless treated in accordance with the relevant laws and regulations to ensure biological safety.
I. Introduction
Liver-Biotech is a national high-tech enterprise focusing on the isolation, cryopreservation and regeneration of primary hepatocytes. Primary duck hepatocytes are isolated from clean grade ducks with high cell purity (>95%). After a variety of tests, the recovery cells are highly viable and polarized (differentiated), and could be widely used in basic life science research and drug discovery.
Ⅱ. Reagents and Materials
-Primary duck hepatocytes (Cat# LV-PDH001)
-Recovery medium (Cat#LV-Rec001)
-Purification medium (provide if needed)
-Plating medium
(Plateable cells are needed. Cat#LV-WEP009)
-Maintenance medium
(Plateable cells are needed. Cat#LV-WEM009)
-Collagen-coated plate
(Plateable cells are needed. Cat #LV-Coated)
-Sterile centrifuge tube of 15ml
-Disposable pipette
-Wide-mouth pipette tip (Cut off the tip of normal pipette and sterilize it.)
-Pipette
-Thermostatic water bath (Preheat at 37℃. Please calibrate the temperature with a thermometer)
-Low-temperature horizontal centrifuge (with a horizontal rotor, it is possible to centrifuge a 15ml centrifuge tube)
-Biosafety cabinet
-37 °C/5% CO2 incubator
-75% alcohol
Ⅲ. Recovery of Suspension Cells
1. Add 10ml recovery medium (Cat#LV-Rec003) to a 15ml centrifuge tube in a biosafety cabinet, pre-warm a thermostatic water bath at 37°C for 20 min, and then transfer the medium to a biosafety cabinet, and pre-warm the plating medium at 37°C.
2. Rapidly transfer the frozen hepatocytes by a liquid-nitrogen thermos from the refrigerated location to a 37°C thermostatic water bath and immerse as much as possible in 37°C water with clockwise horizontal rolling, but must ensure that the cap of cryopreservation tube stays above the water.
3. Thaw the cryopreservation tube for about 90 ~120s until only a small piece of crushed ice floats in it.
4. Sterilize the cryopreservation tube with 75% alcohol. Aspirate the cells with a wide-mouth pipette tip and add them dropwise to a 15ml centrifuge tube containing the preheated recovery medium. (Note: there are some cells left on the cryopreservation tube and the pipette tip, aspirate 1ml recovery medium to rinse. Then, transfer the 1ml recovery medium into the 15ml centrifuge tube). Slightly turn the 15ml centrifuge tube upside down 2-3 times to mix cells well.
5. Centrifuge 100×g of the 15 ml centrifuge tube at low speed, at room temperature for 5min. After the supernatant being removed, resuspend cells with 4ml plating medium, and the viability of hepatocytes was determined by Trypan Blue Exclusion Method. If the viability is more than 70%, it can be plated directly. Otherwise purify the hepatocytes before plating.
6. Cell purification (optional step): Centrifuge hepatocytes suspension at 50×g, at room temperature, for 5 min. Remove the supernatant and resuspend the cells with 2ml of purification medium. Then, carefully adds 1ml plating medium above purification medium along the centrifuge tube wall to the centrifuge tube (Do not stir the lower layer, obvious stratification happens after adding the plating medium). Centrifuge at 800×g at 4°C for 20 min (set maximum acceleration to 9, set minimum deceleration to 1). After centrifugation, aspirate the turbid layer (living cell layer) between the purification medium and the plating medium, and transfer the turbid layer to a new 15ml centrifuge tube. Mix twice volumes of plating medium and the turbid layer in the new 15ml centrifuge tube. After that, centrifuge 100×g of the 15ml centrifuge tube for 5 min at room temperature, remove the supernatant and resuspend the cells with 4ml plating medium.7. Measure the viability and total number of living hepatocytes by Trypan Blue Exclusion Method. It is recommended to repeat 3 times to get an average.
8. Seed the living cells at the density of 1.8×105cells/cm2 onto collagen-coated plate. Shake the plate well and incubate it in a 37℃/5% CO2 incubator.
Ⅳ. Maintenance of Hepatocytes
1. After culturing the hepatocytes for 24 hours, the cells have been well seeded. Remove the plating medium (containing part of the dead cells), add the maintenance medium, and change the culture medium every 2 days.
2. Primary duck hepatocytes have limited proliferation capacity and obvious characteristics of contact inhibition; the differentiation characteristics of hepatocytes and the maintenance time all depend on high-density cell culture.
3. The maintenance time of primary duck hepatocytes in vitro is relatively short. The duration of the experiment is recommended to be completed within 3 days and up to 5 days after plating. It is not recommended to subculture the primary duck hepatocytes.
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. We ensure your after-sales service. There are some objective factors for the failure of the experiment, for example, different laboratory environments, different operating habits and operators’ proficiency, etc. If the operation is not strictly in accordance with the instructions or exceeds the time limit of after-sales service, our company will not provide you with after-sales service. Thank you for your understanding and support.
Validity period of after-sales and original data needed:
Recovery problems: Trypan Blue staining or PI staining should be provided within 24 hours of recovery.
Pollution problems: microscope photographs of differences should be provided within 96 hours of recovery.
Purity problems: immunofluorescence or flow cytometry results should be provided within one month.
Ⅵ. Contact Information
Tel:0755-28284050
Technical Support: 19902901483 (Dr. Zhou)