Cells were slow growing after lentivirus transduction and puromycin antibiotic selection. Any suggestions to grow them better?

Dear Colleague,

I hope this message finds you well. Observing slow cell growth after lentivirus transduction and puromycin selection can be a common issue. This could be due to several factors, including the transduction process, selection pressure, and overall cell health. Below are some detailed and logical suggestions to improve cell growth under these conditions.

Suggestions for Improving Cell Growth Post-Lentivirus Transduction and Puromycin Selection

1. Optimize Lentivirus Transduction

Multiplicity of Infection (MOI):Ensure you are using an optimal MOI for your specific cell type. Too high an MOI can cause cytotoxicity, while too low an MOI may not effectively transduce enough cells. Perform a titration experiment to determine the optimal MOI that balances efficiency and cell viability.

Polyethyleneimine (PEI) or Polybrene:Use polybrene or similar agents to enhance transduction efficiency. A typical concentration is 4-8 µg/mL.

2. Post-Transduction Care

Recovery Period:Allow the cells to recover for 24-48 hours after transduction before applying puromycin selection. This recovery period helps the cells to stabilize and express the resistance gene. Culture the cells in a medium supplemented with 10-20% FBS to promote recovery.

Puromycin Selection:Use the lowest effective concentration of puromycin. Perform a kill curve experiment to determine the minimal concentration that effectively kills non-transduced cells. Gradually increase the puromycin concentration if necessary, to avoid excessive stress on the cells.

3. Enhance Cell Culture Conditions

Medium Optimization:Ensure that you are using a high-quality, nutrient-rich medium appropriate for your cell type. Consider supplementing the medium with additional growth factors or supplements, such as non-essential amino acids, vitamins, or specific growth factors.

Serum Quality:Use high-quality, high-performance FBS or serum. The quality of the serum can significantly impact cell growth and recovery.

Cell Density:Seed cells at an optimal density. Over-confluence or under-confluence can stress the cells. Typically, cells should be 50-70% confluent at the time of transduction.

4. Monitor Cell Health

Mycoplasma Testing:Ensure that your cell culture is free from mycoplasma contamination, as this can significantly impact cell growth and health.

Regular Monitoring:Regularly monitor the cells under a microscope for signs of stress or contamination. Change the medium regularly to remove dead cells and debris.

5. Specific Supplements and Treatments

Antioxidants:Consider adding antioxidants such as N-acetylcysteine (NAC) to reduce oxidative stress during the recovery period.

Conditioned Medium:Use conditioned medium from healthy, rapidly growing cells of the same type. Conditioned medium contains secreted factors that can promote growth and recovery.

Rock Inhibitor (Y-27632):For some cell types, especially stem cells, adding a ROCK inhibitor can improve survival and growth post-transduction.

Example Protocol

Day 0 (Transduction):Seed cells at 50-70% confluency in a 6-well plate. Add lentivirus with an optimal MOI and polybrene (4-8 µg/mL). Incubate for 24 hours.

Day 1-2 (Recovery):Replace the medium with fresh complete medium without puromycin. Allow cells to recover for 24-48 hours.

Day 3 (Selection):Add puromycin at the minimal effective concentration determined from a kill curve (e.g., 1-2 µg/mL). Monitor cell viability daily.

Day 4-7 (Post-Selection):Replace the medium every 2-3 days to remove dead cells and provide fresh nutrients. Monitor cell growth and morphology regularly.

Ongoing Maintenance:Once the resistant population is established, maintain the cells in a medium with the maintenance concentration of puromycin (e.g., 0.5-1 µg/mL). Continue regular monitoring and medium changes.

Conclusion

Improving cell growth after lentivirus transduction and puromycin selection involves optimizing the transduction conditions, providing an appropriate recovery period, using the correct puromycin concentration, and ensuring optimal culture conditions. By following these steps, you can enhance the viability and growth of your cells, leading to more successful experiments.

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