Product Introduction
Catalog:LV-RLSE001 Number of cells:1 million/vial Form: cryopreservation (spot/P1 generation), fresh culture (need to be customized) Inquiries: Please contact on 19902901483 Uses: For research use only |
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Rat sinusoidal endothelial cell
(Can be attachment,Cat# LV-RLSE001)
(For research use only)
Please follow the instructions of the goods,when you use it. If you have any questions, please consult the technicians.
I Introduction
Liver Sinusoidal Endothelial Cell (LSEC) plays an important role in the occurrence of physiological functions and pathological mechanisms of the liver, which has become a hot spot in liver research in recent years. This product is isolated into SPF-grade rats by enzymatic perfusion digestion, retaining the biological activity of cells in vivo. Other liver cells are removed by gradient centrifugation with ultra-high purity (>90%). After detection, there is no contamination of bacteria, fungi, actinomycetes, etc. And the cell viability is high (cryopreservation > 85%, fresh culture >95%)
II Reagents and Materials
- Rat sinusoidal endothelial cell(Cat# LV-RLSE001)
- Resuscitation medium(Cat#LV-Rec001)
- Culture medium of sinusoidal endothelial cell(Cat# LV-RLSEM001)
- Collagen-coated plate(LV-coated)
- Crushed ice and ice box
- Sterile centrifuge tube of 15 ml(Pre-chilled on ice)
- Disposable pipette
- Thermostat water bath(Preheat at 38 °C)
- Wide-mouth pipette tip (The tip of a normal one is cut off and sterilized)
-Pipette
- Refrigerated and horizontal centrifuge (with horizontal rotor,it is possible to centrifuge 15 ml of centrifuge tube)
- Biosafety cabinet
-37 °C/5% CO2 Incubator
-75% Alcohol
III Cell Resuscitation and Plating
Insert the centrifuge tube of 15 ml into an ice box containing with enough crushed ice and sterilize it UV for 15 min. Then, precool centrifuge at 4 °C.
Put the resuscitation medium in crushed ice for fully precooling and put the plating medium into thermostat water bath of 38℃ for fully preheating.
Quickly transfer the frozen cells from the refrigerated position to thermostat water bath of 38℃. Immerse them in as much water as possible at 38°C and shake it horizontally and clockwise, but make sure the cap remains above the water.
Defrost the freezing tube for about 90-120s, until only a small amount of crushed ice floats in it.
Disinfect the freezing tube with 75% alcohol and transfer it to a biosafety cabinet.
Resuspend the cells with a wide-mouth tip (gently blow 2 times) and transfer to a pre-chilled freezing tube of 15 ml.
(Note: Residual cells on the freezing tube and pipette tip, they can be washed with 1 ml of resuscitation medium)
Add pre-chilled resuscitation medium to the cell suspension dropwise, containing 10 ml of r resuscitation medium per 1 ml of cell suspension (Note: When adding, the first 3 ml should be added slowly dropwise and shaken slightly, and the next 7 ml can be accelerated). Finally, mix slightly upside down once.
Centrifuge at 4 °C of 300 × g for 5 min. Remove the supernatant and resuspend with medium.
Pelleted cells should be resuspended with the medium of hepatic sinus endothelial cell and settled to the volume of 4 ml. The survival and total number of cells can be measured by trypan blue exclusion.
Cells are seeded into collagen-coated plates at 3×104cells/cm2. Shake them well and incubate in a 5% CO2 incubator of 37 °C. After 24 h, change the solution.
IV Cell Culture and Passage
1. The cells can be passaged when the degree of fusion reaches 80%.
2. Put the medium, PBS and pancreatin into thermostat water bath of 37℃ for preheating. And wipe it with 75% alcohol before placing in the ultra-clean table.
3. Aspirate the old culture solution and add a small amount of PBS to wash the cells. In addition, add an appropriate amount of pancreatic enzyme so that the amount of pancreatic enzyme can cover the cells. Incubate the cells at 37°C for 2-3 min.
4. Under the microscope, discard the pancreatic enzyme when the intercellular space of the adherent cell becomes larger and the cells tend to be rounded but have not yet floated. After that, fresh hepatic sinus endothelial cell medium should be added and the cell flask should be shaken to terminate the action of pancreatin. Then, carefully blow the adherent cells with a pipette to make a cell suspension (Note:Pay attention to the force of the blowing to avoid generating a large number of bubbles).
5. Centrifuge of 300 × g at room temperature for 5 min. Remove the supernatant and resuspend with medium.
6. Inoculate the cell suspension into a new collagen-coated flask/plate at the density of 3×104cells/cm2 and place it in a 37 °C/5% CO2 incubator. Adherent growth can be observed every other day.
IV 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
V Contact Number
Tel:0755-28284050
Marketing representative: 18129812531 (Manager Deng)
Technical Support:19902901483 (Dr. Zhou)