Lentivirus (LVs) are single stranded RNA (sRNA) viruses, which are a subgroup of the retrovirus family. They can integrate into the host genome in both dividing and non-dividing cells, with a wide range of cell, resulting in long-term expression both in vitro and in vivo. STEP 1.  Prepare target cells
The 24-well plate will be spread at the appropriate cell density (around 0.5×10⁵cells per well) the day before infection. Add 0.5 ml of complete optimal medium (with serum and antibiotics if required), shake gently and incubate at 37 °C with 5 % CO₂ overnight.
Note: The amount of inoculated cells varies slightly depending on the growth rate of the cells. The target cells should be approximately 80% confluent.
STEP 2.  Lentivirus infection of target cells
(1)  Adherent cells
● Prepare lentivirus-containing media: remove the original cell culture media and add the appropriate amount of virus particles to achieve the desired multiplicity of infection (MOI).
Note：Method for calculating the amount of virus particles to be added: (number of cells to be infected × MOI value/titer of virus stock) × 10³ = amount of virus to be added (μl), cells must be fully covered by all media. It is advisable to perform several preliminary tests to determine the desired MOI of lentivirus infection to achieve the highest percentage of successfully infected cells.

Type of petri dish	Surface area	Volume of culture media	Volume of virus solution
96-well plate	0.3 cm2	100 µl	100 µl
48-well plate	0.6 cm2	200 µl	200 µl
24-well plate	2 cm2	500 µl	500 µl
12-well plate	4 cm2	1 ml	500 µl
6-well plate	10 cm2	2 ml	1 ml
T25 flasks	25 cm2	5 ml	2.5 ml

● Add 8 μg/mL polybrene to cell culture media. Which can increase the efficiency of viral infection by 2~10 fold. Please note that polybrene is toxic to some cells. Then incubate the cells with the virus-containing media at 37 °C with 5 % CO² overnight.
● Remove the culture medium and replace with fresh, desired complete medium next day after infection.
(2) Suspension Cell
● According to the experimental conditions determined in the preliminary tests, resuspend the target cells with lentiviral particle solution at a density of 3-5×10⁵ cells/mL and plate the target cells according to a general guideline below.

Type of petri dish	Surface area/cm2	Volume of culture media
48-well plate	0.6 cm2	200 µl
24-well plate	2 cm2	500 µl
12-well plate	4 cm2	1 ml

● There is no need to incubate the cells after plating, add the appropriate amount of virus particles (with 8 μg/mL polybrene it depends on the target cells) to infect target cells at desired MOI. Then shake gently and incubate at 37 °C with 5 % CO₂ for several minutes.
● Transfer the cells into sterile 1.5 ml EP tube and centrifuge for 2 min at 2000g. Remove virus containing medium and resuspend cell pellet with fresh complete culture media, mix gently and continue to culture.
STEP 3.  Check transduction efficiency
The cells are in good condition without a large amount of deaths, especially to ensure that the NC group and the CON group should be the same.
● For viruses with reporter gene (for example GFP), the expression efficiency of fluorescence can be observed by fluorescence microscope 72 h after infection. for viruses with resistance gene (for example Puromycin)
● 48 h after infection, for viruses with reporter gene (for example GFP), the expression efficiency of fluorescence can be observed by fluorescence microscope. For viruses with resistance gene (for example Puromycin), replace with fresh complete culture medium containing appropriate concentration of antibiotic as determined by a killing curve to screen for stable transduction cell lines.
● Generally speaking, if the cell infection efficiency is more than 70%, downstream expression can then be assayed by a number of techniques, including Western blot or RT-PCR.

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