How-to guide for lyovirus use

1. Storage and dilution of lyovirus:
1. Storage of viruses: users receive the virus liquid in a very short period of time to use lyovirus experiments, the virus can be temporarily placed at 4
degrees C to save; Viruses can be stored for more than 6 months at -80 degrees C, but if the virus is stored for more than 6 months, we recommend that you retitration the virus titration before use.
B． Repeated freezing and thawing will reduce the virus titration: each freeze-thaw will reduce the virus titration by 10%, so try to avoid repeated freezing and thawing during the use of the virus, in order to avoid repeated freezing and thawing we strongly recommend that customers receive the virus after each use according to the amount of packing.
2. Dilution of the virus: When the user needs to dilute the virus, remove the virus and place it in an ice bath to melt, use
culture
destination cells with PBS or no
serum
media
(containing serum or double anti-effect virus infection) after mixing and mixing after 4 degrees C (please try to use up within three days), after use.II. Lyovirus for in vitro experiments: infection culture
genogenous cells
and building cells
1. Lyrovirus is affinity for a variety of cells and tissues. Users can learn about the affinity of lyrovirus to your destination cells, the amount of viruses needed for infection complex (MOI value) and in vivo injections before using the lyrovirus provided by Invabio. If there is no relevant literature to support, you can obtain a suitable infection complex (MOI value) (using a 24-well plate to detect the virus's affinity to the intended cell)
2. LYCD purpose cell pre-experiment
(1) LYVIR infection purpose cell pre-experimental note
A. When determining the affinity of trovirus to the intended cells, it is necessary to simultaneously set the cells with high affinity for lyovirus (HEK293T, Hela) as control cells for parallel experiments.
B． In LYC experiments, it can be diluted with a full culture base (for cultured purpose cells);
C． Invabio provides virus units in TU/ml, i.e. the number of biologically active virus particles per milliliter. For example, a virus with a titration of >1X10
8
TU/ml contains at least 1X10
8
bioactive lyrovirus particles per milliliter of viral fluid.
(2) Take the 24-well culture plate as an example, conduct pre-infection experiments on the purpose cells and HEK293T cells
before the experiment, set different infection holes according to different MOI and calculate the amount of virus required according to MOI and cell count, and dilute the virus protosthesis with PBS solution or no serum culture base if necessary
first Day, preparation cells: inoculation of several holes in a 24-well culture plate, inoculation of 3 to 5X10
4
end cells per well, the cell fusion rate at the time of paving is about 50%, the volume of the culture base per hole is 100 μl;
The next day, observe the cell growth state, if the cell state is better, then start the experiment:
1, remove the virus stored at 4 degrees C, centrifuge with a desktop centrifuge for 20 seconds (so that the virus is completely suspended at the bottom of the centrifuge tube); According to the actual situation in the laboratory, the lyptos virus can be accurately calculated according to MOI dilution into the medium, and as far as possible to ensure that the total volume of the medium containing lysovirus is the smallest volume, in order to obtain the best infection efficiency.
2, after the virus is ready, take the cells out of the culture box,
a use the
-lipor
to absorb the exact volume of viral fluid to add the prepared medium
b sucks away the medium in the original
cell culture
vessel (if the cells grow well, the density is appropriate, no change of fluid)
c in The purpose cells and control cells were added to the calculated viral fluid
d mixed and put in the carbon dioxide incubator (37 degrees C, 5% CO
2
) incubation overnight
fill: the state of the pre-infection cells has a great impact on the final infection effect, so it is important to ensure that the cells are in a good growth state before adding the virus.
If lysovirus infection to the intended cell is low, it can increase its infection efficiency by increasing the MOI value, but when the MOI is above 20, we recommend that customers add ploybrene (around 8 μg/ml) to the medium to improve the infection efficiency of the virus.
The third day, change the culture fluid: generally after 24 years of childhood will contain lysovirus culture liquid replaced with normal culture fluid, after infection to observe the state of the cell, if the lyovirus has a significant toxic effect on the cell and affect the cell growth state, can be as short as the virus 4 childhood replacement of fresh culture solution continued culture (recommended in 8-12 hours replacement is appropriate). After the first change of fluid, if the lysovirus has no obvious toxic effect on the cells, the fluid change is cultured according to normal culture conditions.
Day 6, Infection Efficiency Detection: Look at fluorescence in an inverted fluorescence
microscope
to estimate the efficiency of cells at the destination of LY
C
infection;, troviral use precautions
1. It is best to use biosecurity cabinets when operating viruses. Do not turn on the exhaust fan if you are operating the virus using a normal ultra-clean workstable.
. Wear an experimental suit with a mask and gloves when operating a virus.
. Be especially careful not to create aerosols or splashes when operating viruses. If the ultra-clean work station is contaminated with viruses, wipe it clean immediately with 70% ethanol plus 1% SDS solution. Guns exposed to the virus, centrifugal tubes, culture plates, culture fluid should be soaked in 84 disinfectant or 1%SDS overnight and discarded.
. When observing cell infections with a microscope, follow these steps: tighten the culture bottle or cover the plate. Clean the outer wall of the culture bottle with 70% ethanol and look at the photo at the microscope. Before leaving the microscope test bench, clean the microscope test bench with 70% ethanol.
. If centrifugation is required, use a well-sealed centrifuge tube, or centrifuge after sealing with a sealing membrane, and try to use a centrifuge in the tissue culture room.
off your gloves, wash your hands with soap and water. 4. Simple steps for suspended cell infection
1. Centrifugal collection of cells in a 1.5 ml tube according to the amount of cells and dilution of cell precipitation with 100-200ul serum-free culture solution, with cells completely immersed in the medium
2. According to the MOI conversion virus particle count, absorb the viral fluid into the cell, incubate the 1.5 ml tube in a 37 degree C incubator for 30 minutes
3. Suck the mixed solution out of the tube and add it to the petri dish or hole
4. Add a sufficient amount of fresh culture
5.12 hours after changing the fluid
6.96 hours after observing the cell-positive rate