Hi all,
I am doing CRISPR on Hek293T cells. After FACS, the one cell wells do not survive/grow only the wells were 50/100 cells were plated. Any tips to improve the survival rate for the one cell cultures? I use DMEM high glucose with 10 % FBS. The cells are 11-15 µm, so I can not use a 22 µm filter to filter medium from other cells.
Thanks a lot.
For some cells, using conditioned medium (one where the cells have been growing in for a bit) at about 10-20% will help. You can clear any cells in the media by centrifugation, and take the supernatant. No need for filtering.
I also had a problem with the same isssue some time ago, albeit with a different cell line.
For me, the 1. adding conditioned media (50%) as Ron also suggested, 2. coating the wells with a molecules that support adhesion (in my case collagen type I, but also matrigel can be used, depends on the cell line) and 3. keeping cell(s) in a small amount of medium (50ul) for the first week helped me to get single cell clones growing.
Seems that for HEK293 almost anything is good, see the attached file.
Good luck,
Jakub
Dear Esteemed Colleague,
Greetings. I hope this message finds you well and thriving in your research endeavors. Your inquiry regarding the cultivation of HEK293T cells following single-cell sorting is of paramount importance for ensuring the success of your experiments. Single-cell sorting and subsequent culture of such cells require meticulous care to ensure viability and promote growth. Below, I provide a detailed guide designed to optimize the conditions for growing HEK293T cells after single-cell sorting.
1. Preparation Before Sorting
- Cell Condition: Ensure that the HEK293T cells are in optimal condition prior to sorting. This means cells should be healthy, actively growing, and at a low passage number to ensure the best viability and performance post-sorting.
- Media Preparation: Use fresh, complete growth medium tailored for HEK293T cells, typically DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Pre-warm the medium to 37°C to promote immediate cell recovery.
2. Sorting Parameters
- Gentle Conditions: Opt for the gentlest sorting parameters possible to preserve cell viability. High pressures and narrow nozzles can damage cells.
- Single-Cell Purity: Adjust the sorter for maximum purity to ensure that sorted droplets contain a single cell. This may require calibrating the instrument before sorting.
3. Post-Sorting Culture
- Immediate Transfer: Quickly transfer sorted cells into pre-warmed medium to minimize stress and temperature shock.
- Low Attachment Plates: Consider using plates treated for low attachment or coated with poly-D-lysine to prevent cells from attaching too rapidly, which can cause stress in some sorted cells.
- High-Humidity Incubation: Culture the sorted cells in a high-humidity incubator set to 37°C with 5% CO2. This environment helps prevent evaporation and maintains stable conditions conducive to cell recovery and growth.
4. Optimal Culture Conditions
- Reduced Serum Conditions Initially: Some protocols suggest using media with reduced serum (around 5% FBS) immediately after sorting to minimize stress on the cells. After 24-48 hours, when cells have attached and show signs of recovery, the medium can be switched back to the standard 10% FBS formulation.
- Use of Rock Inhibitor: Including a Rho-associated protein kinase (ROCK) inhibitor in the culture medium immediately post-sorting can enhance survival of single cells by preventing apoptosis.
- Monitoring and Subculturing: Carefully monitor cell growth and morphology. When cells reach an appropriate density, typically within 7-14 days post-sorting, they can be gently trypsinized and expanded under normal culture conditions.
5. Troubleshooting
- Low Viability: If viability is low post-sorting, consider optimizing the sorting parameters, the composition of the sorting buffer, and the immediate post-sort recovery conditions.
- Slow Growth: Single cells, especially after sorting, may take longer to divide and establish a colony. Patience is key, but if growth is exceptionally slow, verify the incubator settings and the freshness and composition of the media and supplements.
Adhering to these detailed tips and maintaining a meticulous approach to the post-sorting care of HEK293T cells will significantly enhance the likelihood of successful culture from single-cell sorts. It is crucial to adapt these guidelines as necessary to fit the specific requirements of your experimental setup and the unique characteristics of your cell line.
Should you have any further questions or require additional insights into cell culture techniques, please do not hesitate to reach out. I am here to support your scientific exploration and contribute to the advancement of your research.
Warm regards.
Perhaps this protocol list can give us more information to help solve the problem.
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