Hello everyone,
I am currently trying to assess cell viability on a cell culture treated with different drugs. No problems with DAPI staining, but I am experiencing some problems with PI.
I tried different concentrations (from 1 mg/ml to 0.01 ug/ml) and times of incubation (from 1 to 10 minutes). I am getting diffuse staining of cells, with the nucleus and nucleoli being particularly evident (but also the cytoplasm is stained). If I try with higher dilutions I only get faint nuclear fluorescence, but I cannot see any nucleus properly stained (as if there are no dead cells, which seems quite weird).
Cells have been previously fixed with paraformaldehyde and then kept in PBS. The PI was a powder that I diluted in PBS. Is it possible that PI might be expired or not properly conserved?
Did any of you experience some of these problems?
Thank you
I've stained with PI only bacterial cells and the staining was always strong. We kept the stock solution at -20. Maybe membranes of your cells are intact? I would try to permeabilize the cells for the control PI staining. Usually, PI stock solution should be prepared in water, but I don't think PBS might be a problem.
Irshad Sharafutdinov thank you. PI in my case should stain only the nucleus of dead cells. But it stains everything or, at higher dilutions, nothing...
Dear Ilaria!
https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/propidium-iodide
1 Propidium iodide dye exclusion assay
The propidium iodide dye exclusion assay is performed in a Krebs–Ringer–HEPES buffer (KRH: 115 mM NaCl, 5 mM KCl, 1 mM KH2PO4, 1.2 mM MgSO4, 2 mM CaCl2, and 25 mM HEPES at pH 7.4) containing 30 μM propidium iodide.
https://cancerres.aacrjournals.org/content/canres/49/14/3776.full.pdf
At the time appropriate for assessment of viability, an aliquot (usually 2 ml) of each culture is placed in a test tube, and then FDA is added to each test tube to achieve a final concentration of 0.5 Mg/ml- The tubes are incubated at room temperature in the dark for 30 min, and then PI in isotonic saline is added to each tube to achieve a final concentration of 50mkg/ml. The tubes are then immediately placed into ice, and kept ice-cold during flow cytometric analysis. Viability is assessed by determining
As correctly noted, PI stains nuclei, DNA, and therefore the cells must be permeable to the dye (have a damaged membrane). Maybe your drugs do not harm the cell membrane, but have a specific effect through receptors. Try other drugs with a strong cytotoxic effect (for example, chemotherapy).
We also are performing a cell culture in which first we fix cells with paraformaldehyde, in which we clean afterwards using PBS, after the cleaning we stain with PI at a concentration of 10ug/ml, using a intermediate solution stock stored at 2-6ºC (fridge) our problem is how much time should be the PI on the sample? As sometimes we see artefacts in our preparations.
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