Product Introduction
Kupffer Cell
(Plateable, Cat# LV- Kup001/2/3/4)
(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
Kupffer cell plays an important role in the physiological function and pathological mechanism of the liver, and have become a hot topic in liver research in recent years. This product is isolated by collagenase perfusion digestion to preserve the biological activity of the cells in vivo, and other liver cells are removed by Percoll gradient centrifugation, and then subjected to differential attachment for ultra-high purity (>80%). After testing, there is no contamination by bacteria, fungi, actinomycetes, etc., and the cell viability is high (cryopreservation > 80%, fresh >95%). The company can customize the primary hepatocytes to meet the needs of different customers.
Ⅱ. Reagents and Materials
- Kupffer cell (Cat# LV- Kup001/2/3/4)
- Kupffer cell culture medium (Cat# LV-KuPM001)
- Culture plate (TC-treated)
- 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 38℃.)
- 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. Preheat Kupffer cell culture medium well in a 38°C water hermostatic bath.
2. Rapidly transfer the frozen Kupffer cells by a liquid-nitrogen thermos from the refrigerated location to a 38°C thermostatic water bath and immerse as much as possible in 38°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. Centrifuge 300×g of the 15 ml centrifuge tube at 4℃ for 5min. After the supernatant being removed, resuspend cells with plating medium.
5. The pelleted cells are resuspended with Kupffer cell culture medium and bring to a volume of 4 ml. Use Trypan Blue Exclusion Method to determine cell viability and total amount.
6. Seed the living cells at the density of 3×104cells/cm2 onto TC-treated culture plate. Shake the plate well and incubate it in a 37℃/5% CO2 incubator, change the culture medium after 24h.Ⅳ. Cell Culture and Subculture
1. Cells can be passaged when they are 80% confluent.
2. Preheat the culture medium, PBS and pancreatic enzyme into a 37℃ water bath in advance, wipe it with 75% alcohol, and then put it into the ultra-clean table.
3. Aspirate the old culture medium, add a small amount of PBS to rinse the cells, and an appropriate amount of trypsin, so that the added trypsin can cover the cells. Incubate at 37°C, and observe under the microscope for about 2~3min.
4. When the space between the plateable cells becomes larger and the cells become round but not yet floating, discard the pancreas. Add fresh medium, shake the cell bottle, stop the pancreatic enzyme. Use a straw carefully to blow the plateable cells to make cell suspension (control the intensity of blowing, to avoid a large number of bubbles).
5. Centrifuged at 300×g for 5min at room temperature, remove the supernatant and resuspend it with Kupffer cell culture medium.6. Seed the cell suspension at the density of 3×104cells/cm2 onto a new TC-treated culture plate and incubate it in a 37℃/5% CO2 incubator. Observe the growth of plateable cells next day.
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)