Dear friends Andrew said "Andrew D. Wells, Ph.D." <adwells@mail.MED.UPENN.EDU> Subject: Re: TUNEL Assay >I have a question about the Tunel assay using flow cytometry. I treated my >cells ( adherent cell line) with agents, Actinomysin D, Ceramide, >Campothecin, before staining with dUTP-FITC (kit from boehringer mannhein) >to detect apoptosis.What does mean when the label sample(negative sample) is >higher( histogram peak) than positive sample (TdT sample). >I would appreciate any help anyone can give. To his question Andrew got an answer concerning cell autofluorescence. "Although its unlikely that the following point will explain (all) your problem, I thought it would be worthwhile to mention to you and the mailing group that, in my experience, some drugs that one might use to induce apoptosis are retained in the cells and can act as fluorochromes when hit by the laser; i.e., your drug-treated, non-TdT-treated cells can fluoresce quite brightly in FL1 and 2. Again, this can't explain every case of high background in TUNEL, but its important to be cognizant of this situation. And why your TdT-treated would be less fluorescent than your control is not clear to me (quenching of the background by the FITC?). Hope this helps, AW The point is that normal non-treated cells exhibit at least two type of characteristic autofluorescence. One in the blue range which concern NAD(P)H fluorescence and the other one concerning the flavoproteins in the green and orange green range. Any type of cell induced to apoptosis undergo a characteristic change in both autofluorescence The NAD(P)H fluorescence essentially originating from mitochondria decrease as the mitochondrial membrane potential do. And the flavoprotein fluorescence increase slightly. In case of cell with an important cytoplasm over nuclei ratio, the treated cells may have an significatively increased Fl1 (sometime Fl2) fluorescence which can be the reason of the observed problem you submitted. This could disturb partly the measurement of the tdT assay. I do not see any other major problem.... Sincerely yours Patrice \ | / (o o) \ | / _______________oOo_____oOo_____oOo_ (o o)__oOo________________ Dr. Petit Patrice X. Institut Cochin de Génétique Moléculaire INSERM U129 - CHU Cochin Port-Royal 24, rue du Faubourg Saint-Jacques F-75014 Paris, France. Tel: 33 01 44 41 24 11 Fax: 33 01 44 41 24 21 E-mail: pxpetit@icgm.cochin.inserm.fr
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