How to find the best MOI value of slow virus

MOI (multiplicity of infection), the multiplicity of infection, refers to the ratio of virus to cell number at the time of infection. It is generally believed that MOI is a ratio, there is no unit, in fact, its implied unit is pfu number/cell. However, for some viruses, such as AAV viruses, it is not possible to express the number of viruses with pfu, but to use TU, IU, viral particles (vp) or vector genome (vg) to indicate the number of viruses.

The Lentivirus vector is a single-stranded RNA viral vector based on HIV-1 (human immunodeficiency type I virus) that infects dividing or non-dividing cells and efficiently integrates foreign genes into the host chromosome . Low immunogenicity is widely used in various in vitro cell experiments.

• Common cellular MOI reference value (lent virus)

• Lentiviral MOI value exploration step

• Pre-experiment information preparation

• MOI gradient setting

Two gradients are set before and after the reference MOI value, and each gradient is at least 2 times different. For example, if a cell line is found in the literature, the MOI application is 10, and the MOI gradient is set to 2.5-5-10- 20-40.

Calculate the amount of virus you need to add based on the formula:

MOI value = virus titer (TU / mL) × virus volume (mL) / number of cells

• Paving cells

1×10 ⁴ cells/well were plated in a 96-well plate, and the medium was adjusted to a volume of 100 μL/well;

• Lentivirus infection

Based on the calculated virus volume, lentiviruses were added one by one and fresh medium was replaced the next day of infection.

(5) Determine the MOI range

a. Lentivirus with fluorescent label: observe fluorescence

72 hours after infection, the cell state and fluorescence were observed under a fluorescence microscope, and the wells with better cell state and higher fluorescence number were determined, which corresponded to a suitable MOI value.

• Lentivirus without fluorescent label: resistance screening (puromycin/blasticidin, etc.)

Check the concentration of the corresponding resistance in the corresponding cell line, add the drug at 72 hours after infection, culture for 3-5 days, observe under the microscope, select the well with higher cell survival rate and better cell state, correspondingly suitable The MOI value.

(6) Reduce the MOI range for a second exploration (additional steps)

For more accurate MOI values, the MOI gradient can be set within the MOI range determined in step (5). The experimental method is the same as steps (3)-(5). Example:

• Frequently asked questions in lentivirus infection

(1) How to improve the infection efficiency of lentivirus?

A good growth state of the cells is a guarantee for achieving high infection efficiency. If necessary, ADV-HR can be added to the infection to increase the infection efficiency of the lentivirus.

(2) What is the concentration and method of use of ADV-HR?

The helper agent ADV-HR significantly increased the efficiency of lentivirus infection with a dose- and time-dependent effect. However, higher concentrations of ADV-HR are cytotoxic, affecting cell status and infection efficiency. We recommend that you perform a pre-experiment of the ADV-HR concentration gradient in the cells of interest. Our experiments in HEK293 showed that when ADV-HR was used at a concentration of 1.0 × 10 ^-2 mg/mL or less, the cell state was good.

(3) Why do a large number of black spots appear in cells after lentivirus infection and affect cell growth?

After eliminating bacterial and fungal contamination, the black spots in the cells are usually cell debris, which causes many reasons for cell breakage. However, after lentivirus infection, there are usually two reasons for cell breakage: a. Lentiviral use is excessive. Or too few cells; b. Mycoplasma contamination.

Mycoplasma contamination is ignored by many laboratories because mild mycoplasma contamination does not affect cell growth and proliferation. However, mycoplasma is easy to erupt after the virus infects cells, so a large amount of cell debris will appear. We recommend that when using viral products, you should first eliminate the contamination of cells, cultures and mycoplasma in the culture environment to save your precious time.

(4) How should the lentivirus be saved?

It is recommended that you dispense the drug as soon as it is received and store the virus in liquid nitrogen or -80 °C. Do not freeze and thaw repeatedly! In the case of no freezing and thawing, the storage environment of -80 ° C can be guaranteed for 6 months. If the storage conditions are exceeded, the quality control should be re-examined.

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