How can Ì fix and permeable a cell line expressing GFP for proliferation assay using ki-67?

Performing a proliferation assay with Ki-67 in GFP+ Jurkat cells is a valuable approach to study cell cycle dynamics. However, the issue you’ve encountered with GFP loss during the permeabilization step is common. Let’s address this problem and explore potential solutions:

GFP Leakage During Permeabilization:GFP, being a relatively small protein, can indeed leak out of cells during permeabilization. The permeabilization step (using Triton X-100) disrupts cell membranes, allowing the dye to escape. Consequently, the percentage of GFP+ cells decreases, affecting accurate analysis.

Strategies to Preserve GFP Signal: a. Optimize Permeabilization Conditions:Reduce the concentration of Triton X-100 or the duration of permeabilization. Test different conditions to find a balance between preserving GFP signal and achieving sufficient permeabilization for Ki-67 staining. b. Use Alternative Permeabilization Methods: Try alternative permeabilization agents (e.g., saponin) that may be gentler on cell membranes. Saponin selectively permeabilizes without causing significant GFP leakage. c. Fixation Before Permeabilization: Fix the cells first (as you’re currently doing) to stabilize GFP. Then perform permeabilization. This order may help retain GFP within the cells. d. Use a Different Antibody for Ki-67 Detection: Instead of directly conjugated anti-Ki-67 antibodies, use unconjugated antibodies followed by a secondary antibody. This way, you can avoid permeabilization altogether during Ki-67 staining. e. Consider Live-Cell Ki-67 Staining: Use a live-cell Ki-67 staining protocol. This method avoids fixation and permeabilization, preserving GFP signal. However, it requires careful optimization and validation.

Positive Control and Validation:Include a positive control (e.g., cells treated with a known cell cycle inhibitor) to validate your Ki-67 staining. Ensure that the Ki-67 staining pattern aligns with expectations.

Literature and Protocols:Search for published protocols or literature where researchers have successfully combined GFP and Ki-67 staining. Learn from their methods and adapt them to your specific cell line and experimental conditions.

Collaborate and Share Experiences:Reach out to colleagues or collaborators who work with similar assays. Share your experiences and learn from theirs.

Remember that optimizing protocols often involves trial and error. Be patient, systematically test different conditions, and document your findings. With persistence, you can achieve reliable GFP and Ki-67 staining without compromising GFP signal.

Shafieeh Mansoori

Thanks.

I will try to optimize concentration of tritonx-100 and duration of permeablization.

Also I will test saponin, although it has not recommended for GFP+ cells.

Adam Figarski

GFP fluorescence is killed chemically by formaldehyde/paraformaldehyde/formalin, it is a common problem.

You may stain fixed cells with antibody against GFP (Preferably in green range of colour like ALEXA 488) and separate colour of antibody for Ki -67. I doubt staining on unfixed/unpermeabilised cells will work for Ki-67. What stops You to perform 2 cytometries, one for GFP on unfixed cells and other part of the same sample for Ki67 on fixed cells ?

Shafieeh Mansoori

Adam Figarski

thanks for your reply.

I saw some recommendations for using the paraformaldehid (PFA) to fix GFP+ cells because PFA lead to cross linking of intracellular proteins include GFP and therefore it prevents from coming out of GFP (look the attached file).

about your question: I have to check proliferation assay for GFP+ cell. since permeblizing is nesessary for KI-67 staining, however I have to fix and permeablize GFP+ cells. I have to do analysis with flowjo and at first draw gate GFP+ cells in FL1 channel and then draw FL2(KI-67) gate in child of FL1.

I hope I have explained it clearly.

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