First, the storage and dilution of lentivirus:
1. Storage of virus: After receiving the virus solution, the user can use the lentivirus for experiment in a short time. The virus can be temporarily stored at 4 °C. If it needs to be stored for a long time, please place it at -80 °C. Tube and seal with a sealing membrane)
Note: A. Virus can be stored at -80 ° C for more than 6 months; but if the virus is stored for more than 6 months, we recommend re-tipping the virus titer before use.
B. Repeated freezing and thawing will reduce the virus titer: each freeze-thaw will reduce the virus titer by 10%; therefore, we should try to avoid repeated freezing and thawing during the use of the virus. To avoid repeated freezing and thawing, we strongly recommend that customers follow the virus after receiving it. Each time the amount of use is divided.
2. Dilution of the virus: When the user needs to dilute the virus, please take out the virus and dissolve it in an ice bath. Mix the cultured cells with PBS or serum-free medium (containing serum or containing double antibody without affecting the virus infection). Store at 4 °C (please use up within three days) and use after dispensing.
Second, lentivirus for in vitro experiments: infection of primary cells and lineage cells
1. Lentiviruses have different affinities for various cells and tissues. Before using the lentivirus provided by Invabio, users can consult the relevant literature to understand the relative affinities of the lentivirus to your target cells, the multiplicity of infection (MOI value), and The amount of virus required for in vivo injection. If there is no literature support, a suitable multiplicity of infection (MOI value) can be obtained by infection pre-experiment (using a 24-well plate to detect the affinity of the virus for the target cell)
2. Pre-experiment of lentiviral infection of the target cells
1 Pre-experimental notes on the target cells of lentivirus infection
A. When measuring the affinity of a lentivirus to a cell of interest, it is necessary to simultaneously set a (HEK293T, Hela) cell with higher lentivirus affinity as a control cell for parallel experiments.
B. In the lentiviral infection experiment, it can be diluted with complete medium (for culturing cells of interest); in theory, complete medium containing serum, double antibody or other trophic factors does not affect the infection efficiency of lentivirus.
The unit of virus provided by C. Invabio is TU/ml, which is the number of biologically active virus particles per ml. For example, the virus titer is >1X108 TU/ml, that is, at least 1×108 biologically active lentiviral particles per ml of virus solution.
2 Taking 24-well culture plates as an example, pre-experiment of infection of target cells and HEK293T cells
Different infection wells were set according to different MOI before the experiment, and the required amount of virus was calculated according to MOI and cell number. If necessary, the virus stock solution could be diluted with PBS solution or serum-free medium.
On the first day, prepare cells: inoculate several wells in a 24-well culture plate, inoculate 3~5×104 cells of interest in each well, and the cell fusion rate is about 50% when plating, and the medium volume per well is 100 μl; The degree of cell fusion at the time of infection is about 70%.
The next day, observe the cell growth state, such as the cell state is better to start the experiment:
1. Remove the virus stored at 4 °C and centrifuge it for 20 seconds in a benchtop centrifuge (so that the virus is completely suspended at the bottom of the tube). If it is frozen at -80 °C, the virus needs to be melted on ice. It is also possible to dilute the lentivirus accurately calculated according to the MOI into the medium according to the actual situation of the laboratory, and to ensure that the total volume of the obtained lentivirus-containing medium is as small as possible in order to obtain the best infection efficiency. .
2. After the virus is ready, take out the cells from the incubator.
a Pipette the exact volume of virus solution into the prepared medium
b Aspirate the medium in the original cell culture vessel (if the cells grow well and the density is appropriate, do not change the solution)
c Add the calculated virus solution to the target cells and control cells respectively
d Mix and place in a carbon dioxide incubator (37 ° C, 5% CO 2 ) overnight
Note: The state of the cells before infection has a great influence on the final infection effect, so be sure to ensure that the cells are in good growth state before adding the virus.
If the lentivirus is less efficient in infecting the target cells, increase the MOI value to increase its infection efficiency. However, when the MOI is higher than 20, we recommend that the customer add floybrene (about 8 μg/ml) to the medium to improve. The efficiency of infection of the virus.
On the third day, the culture medium is changed: the culture medium containing the lentivirus is usually replaced with a normal culture solution after 24 hours; the state of the cells is observed after the infection, and if the lentivirus has a significant toxic effect on the cells and affects the cell growth state, it can be the shortest. Continue to culture after replacing the fresh medium with the virus for 4 hours (recommended to be replaced in 8-12 hours). After the first liquid exchange, if the lentivirus has no obvious toxic effect on the cells, the medium is changed according to the normal culture conditions.
On the sixth day, infection efficiency test: fluorescence was observed by inverted fluorescence microscope to estimate the efficiency of lentivirus infection of the target cells; how to select the vector could not carry Marker Gene due to the large target gene, and can be detected by Real-time RT-PCR. The expression of the target gene was used to evaluate the infection efficiency.
Third, the use of slow virus precautions
1. It is best to use a biosafety cabinet when operating the virus. Do not open the exhaust fan if you are using a normal clean bench to operate the virus.
2. Wear a lab coat with a mask and gloves when handling the virus.
3. Be especially careful not to create aerosols or splashes when handling the virus. If the ultra-clean workbench is contaminated with viruses during operation, immediately wipe it off with 70% ethanol and 1% SDS solution. The tip of the virus, the centrifuge tube, the culture plate, and the culture solution should be soaked in 84 disinfectant or 1% SDS overnight and discarded.
4. Observe the cell infection with a microscope. Follow these steps: Tighten the flask or close the plate. The outer wall of the culture flask was cleaned with 70% ethanol and photographed at the microscope. The microscope bench was cleaned with 70% ethanol before leaving the microscope bench.
5. If centrifugation is required, use a well-sealed centrifuge tube, or seal with a parafilm, and use a centrifuge in the tissue culture chamber whenever possible.
6. After removing the gloves, wash your hands with soap and water.
Fourth, simple steps of suspension cell infection
1. Centrifuge the cells in a 1.5 ml tube according to the amount of cells and dilute the cell pellet with 100-200 ul of serum-free medium, subject to complete immersion of the cells in the medium.
2. Calculate the number of virus particles according to MOI, add the virus solution to the cells, and incubate the 1.5 ml tube in a 37 ° C incubator for 30 minutes.
3. Aspirate the mixed solution from the tube into the culture dish or into the well.
4. Add enough fresh medium
5. Change the liquid after 12 hours
6. Observed cell positive rate after 96 hours
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