Lentivirus (LV) In Vitro Infection Cell Experiment Guide: Procedures, Precautions, and Troubleshooting Tips

1. Core Process: Standardized Steps for Lentivirus In Vitro Infection Cell

Step 1: Pre-infection Preparation (Cells + Virus)

🔹Target Cell Preparation:
Select target cells in the logarithmic growth phase (optimal condition, highest transduction efficiency) and seed them into suitable culture containers (6-well plates/12-well plates, as required). Control the seeding density to ensure the cells reach 40%-60% confluency at the time of infection (too high a density may cause competition for nutrients, while too low a density may affect infection efficiency). Culture cells overnight in antibiotic-free complete medium and observe their condition the next day (only proceed if there is no contamination and cells are highly viable).

Step 2: In Vitro Infection Operation (Core Steps)

Figure 1: Schematic of Lentivirus Vector Production and Target Cell Transduction
 

Step 3: Selection and Verification (Essential for Constructing Stable Cell Lines)

🔹Fluorescence Observation:

48-72 hours after infection, observe the proportion of fluorescence-positive cells using a fluorescence microscope to preliminarily judge the infection efficiency (≥70% positive cells is ideal).

🔹Drug Selection:

If constructing a stable cell line, add the corresponding selection drug (e.g., puromycin 2-5 μg/mL) to the culture medium according to the MTC value determined in the pre-experiment. Change the drug-containing medium every 2-3 days and continue selection for 7-14 days until all cells in the blank control group die, while the experimental group cells grow normally.

🔹Clonal Culture (Optional):

To obtain a homozygous stable cell line, use the limiting dilution method to seed the selected cells into a 96-well plate. After single-clone culture, select clones with uniform fluorescence and good growth conditions for further expansion.

🔹Expression Verification:

Use qPCR (to detect gene transcription levels), Western Blot (to detect protein expression levels), or flow cytometry to verify stable expression of the target gene. Once the cell line is confirmed to be qualified, store in liquid nitrogen for backup.

Figure 2: Schematic of Constructing Stable Cell Lines Using Lentivirus

 

II. Exclusive Precautions for In Vitro Infection: Key Pitfalls to Avoid
🔹Cell Status Control: Only use target cells in the logarithmic growth phase and free from contamination for infection. Senescent, apoptotic, or contaminated cells will significantly reduce transduction efficiency. Prior to infection, cell viability can be assessed using Trypan Blue staining.

🔹MOI Optimization: Different cells show significant variability in their sensitivity to lentivirus. For the first experiment, set up a gradient of MOI values (e.g., 5, 10, 20, 50) to identify the optimal MOI, balancing both infection efficiency and cell toxicity.

🔹Polybrene Usage Cautions: Some cells (e.g., neurons, stem cells) are sensitive to Polybrene and may experience cytotoxicity. For these cells, Polybrene should either be omitted or used at a low concentration (2-4 μg/mL) with shortened incubation times.

🔹Aseptic and Antibiotic Usage: Strict aseptic techniques should be followed throughout the experiment. Use antibiotic-free culture medium the day before and during the infection stage. Antibiotics may interfere with the virus binding to the cell surface receptors, alter membrane permeability, induce cell stress responses affecting cell viability, or even create potential antagonistic effects with reagents like Polybrene, thus lowering transduction efficiency. If contamination is a concern, antibiotics can be added during the selection phase.

🔹Virus Usage Guidelines: After thawing, lentivirus should be stored on ice, avoiding exposure to room temperature for extended periods (more than 1 hour can lead to reduced virus activity). Any remaining virus solution should be aliquoted and stored at -80°C, avoiding repeated freeze-thaw cycles.

 

III. Common Problems and Solutions for In Vitro Infection: Quick Troubleshooting