Has anyone successfully sorted GFP positive CRISPR transfected HepG2 single clones using FACS or serial dilution?

29 April 2016 4 8K Report

Has anyone successfully isolated GFP expressing CRISPR transfected HepG2 single clones?

Currently, I am trying to isolate single clones, post-transfection of a GFP expressing plasmid within HepG2 isogenic population. I have attempted a single clone isolation by both dilution and FACS in several 96 well plates.

Following the stress from the FACS sort, the cells do not survive long enough for clonal expansion. I have attempted to grow GFP positive cells in a T25 and follow this with dilution to isolate a single clone. However, I still remain unsuccessful to generate a population for expansion.

Does anyone have any suggestions or ideas?

Michela Perego

it sounds to me like a cell line issue. it is relatively easier obtaining single-cell clones from tumor cell lines, could be more complicated with normal cells, especially differentiated. If the sorting itself is not affecting viability, a general suggestion is to add little more serum to the culture or some factors known to stimulate proliferation or health of your cells, but growth factors could influence your experiments too, but maybe some factors for few days (then removes as soon as you have a small clone) could be a solutions. another option is trying with another cell line, maybe more successful. Good luck

Eric Haertel

Hey Stacey,

have you tried using a 'wider' sorter nozzle when sorting your cells?

This would reduce the shear stress in the flow stream and increase cell viability, especially for hepatocytes, which prefer a gentle touch. ;)

Also you can sort them directly into their preferred medium (incl. serum and supplements etc) while your sort is running (for probably quite some time).

Hope this helps.

Ariane Neumann

Hey Stacey,

I'm also using HepG2 cells und noticed that it's not possible to generate single clones in 96 well plates. HepG2 cells are very sensitive and can only grow if there are other cells around.

I'm seeding the cells in a very high dilution into 10cm dishes (after resuspending the cells, you have to needle them to really get single cells!) and growing them in conditioned medium. As soon as there a colonies forming and you can see that the colonies are deriving from one cell, you can pick them and transfer them to 96 well plates.

This seems to work quite good in my case!

Yu Wa

Dear Colleague,

I hope this message finds you well. Sorting GFP-positive CRISPR-transfected HepG2 single clones can be a challenging but rewarding process. Both Fluorescence-Activated Cell Sorting (FACS) and serial dilution are commonly used methods for isolating single clones. Here, I provide a detailed and logical approach to both methods.

Sorting GFP-Positive CRISPR Transfected HepG2 Single Clones

Method 1: FACS (Fluorescence-Activated Cell Sorting)

Transfection and Preparation:Transfection: Transfect HepG2 cells with your CRISPR-GFP construct using a suitable transfection reagent (e.g., Lipofectamine 3000). Optimize transfection conditions to achieve high efficiency. Recovery: Allow cells to recover for 48-72 hours post-transfection to ensure sufficient GFP expression.

Preparation for FACS:Cell Harvesting: Gently harvest the cells using trypsin or an alternative detachment method. Avoid over-trypsinization to maintain cell viability. Resuspension: Resuspend the cells in FACS buffer (e.g., PBS with 2% FBS and 1 mM EDTA) to a concentration of 1-5 million cells/mL. Filtering: Pass the cell suspension through a 40 µm cell strainer to remove clumps.

Sorting:FACS Instrument: Use a FACS machine equipped with a 488 nm laser for GFP detection. Gating: Set up appropriate gates to select GFP-positive cells while excluding dead cells and debris. Use forward scatter (FSC) and side scatter (SSC) to gate on live cells. Single-Cell Sorting: Sort GFP-positive cells into a 96-well plate containing conditioned medium (medium from cultured HepG2 cells, filtered and supplemented with 10% FBS) to enhance survival.

Post-Sorting Care:Incubation: Incubate the sorted cells at 37°C in a CO2 incubator. Monitor for cell growth and GFP expression. Expansion: Once single clones are visible, expand them to larger wells for further analysis.

Method 2: Serial Dilution

Transfection and Preparation:Follow the same transfection and recovery steps as described for FACS.

Serial Dilution:Cell Harvesting: Harvest the transfected cells gently. Dilution: Prepare a cell suspension and perform serial dilutions to achieve approximately one cell per well in a 96-well plate. Typically, a dilution series of 1:2 or 1:4 works well. Plating: Plate the diluted cells in 96-well plates, with each well containing conditioned medium.

Isolation and Expansion:Incubation: Incubate the plates at 37°C in a CO2 incubator. Check wells periodically for the presence of single-cell clones. Identification: Identify wells with single GFP-positive clones using a fluorescence microscope. Expansion: Once single clones are identified, carefully expand them to larger culture wells for further growth and analysis.

Tips for Success

Optimize Transfection Efficiency: High transfection efficiency will increase the number of GFP-positive cells, making sorting more efficient.

Ensure Cell Viability: Handle cells gently during harvesting and sorting to maintain high viability.

Use Conditioned Medium: Conditioned medium can improve the survival and growth of single-cell clones.

Verify Clones: After isolation and expansion, verify the presence and correct editing of the CRISPR target in GFP-positive clones using PCR, sequencing, or Western blotting.

Conclusion

Both FACS and serial dilution are effective methods for isolating GFP-positive CRISPR-transfected HepG2 single clones. FACS offers precise and rapid isolation of single cells, while serial dilution is a simpler method that does not require specialized equipment. The choice of method depends on available resources and specific experimental needs.

Check out this protocol list; it might provide additional insights for resolving the issue.

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