Product Information

Instructions for Use

Before starting formal experiments, a preliminary lentiviral transduction experiment should be performed using the target cells.

This preliminary experiment serves two purposes:

1. To determine whether the lentivirus can efficiently transduce the target cells.

2. To optimize the transduction conditions, particularly the appropriate multiplicity of infection (MOI).

Lentiviruses carrying fluorescent markers are commonly used for preliminary optimization. The recommended procedures are described below.

I. Lentiviral Transduction in 96-Well Plates

1. Cell Seeding

One day before transduction, seed approximately 1 × 10⁴ target cells per well in a 96-well culture plate. It is recommended to use at least five wells for preliminary testing.

At the time of transduction, the cell confluency should be approximately 50–60%.

2. Ten-Fold Serial Dilution of Lentivirus

Perform a 10-fold serial dilution of the lentiviral stock.

For example, when using a lentivirus with a titer of 1 × 10⁸ TU/mL:

• Add 10 μL of lentiviral stock to 90 μL of basal culture medium and mix thoroughly.

• Transfer 10 μL of the diluted virus to another 90 μL of basal culture medium and mix thoroughly.

Avoid generating bubbles during mixing.

To improve transduction efficiency, polybrene may be added at a final concentration of 5–10 μg/mL.

3. Lentiviral Transduction

Add 10 μL of each viral dilution to the corresponding wells of the 96-well plate.

For each dilution gradient, add 10 μL of the diluted virus to three replicate wells.

Under the conditions described above, the three dilution gradients correspond to approximate MOI values of:

• MOI = 100

• MOI = 10

• MOI = 1

If the viral titer is lower than 1 × 10⁸ TU/mL, increase the volume of lentivirus accordingly to achieve the desired MOI range.

Additional dilution gradients may be used to further optimize the MOI and identify the most suitable transduction conditions for the target cells.

4. Incubation and Evaluation

Culture the cells in a humidified incubator at 37°C with 5% CO₂.

After 48–72 hours, evaluate fluorescent protein expression.

For cells with slower growth rates or lower metabolic activity, the observation period may be extended as appropriate.

Determine the optimal viral dilution based on the overall cell growth status and fluorescence expression level. The corresponding viral concentration can then be used as a reference for subsequent experiments.

II. Lentiviral Transduction in 6-Well Plates

1. Cell Seeding

Seed approximately 1 × 10⁵ cells per well in a 6-well culture plate.

Culture the cells overnight in a humidified incubator at 37°C with 5% CO₂ for transduction the following day.

At the time of transduction, the cell confluency should be approximately 50–60%.

2. Calculation of the Required Lentiviral Volume

Calculate the required lentiviral volume according to the following formula:

Lentiviral volume = (MOI × number of cells) / viral titer

If the viral titer is unknown, a commonly used starting condition is:

• 80–100 μL concentrated lentivirus

• 1 mL culture medium

• 7.5 μL polybrene

This corresponds to a final polybrene concentration of approximately 5–10 μg/mL.

3. Lentiviral Transduction

Remove the existing culture medium from the cells.

Add the prepared lentivirus-containing medium supplemented with polybrene to the cells and gently mix.

Return the plate to the incubator and continue culturing.

4. Post-Transduction Culture

After overnight incubation, add 1 mL of fresh complete culture medium and continue culturing.

Monitor fluorescent protein expression as appropriate.

Precautions

1. Avoid repeated freeze-thaw cycles. Repeated freezing and thawing may reduce lentiviral activity.

2. Wear appropriate personal protective equipment (PPE). Wear a laboratory coat, disposable gloves, and a face mask as required. After completing the experiment, disinfect gloves appropriately and wash hands thoroughly.

3. Perform all lentivirus-related procedures in an appropriate biological safety cabinet.

4. Handle lentiviral materials with care. If lentiviral solution contaminates the work surface, immediately disinfect and clean the area using an appropriate disinfectant according to your institution's biosafety procedures.

5. Properly decontaminate all materials that have come into contact with lentivirus before disposal, including pipette tips, centrifuge tubes, culture plates, and culture media.

Research Use Only

This product is intended for research use only (RUO) and is not intended for diagnostic, therapeutic, or clinical applications.

The information provided above is for reference only. Users should follow applicable institutional biosafety requirements and relevant regulations when handling lentiviral materials.