Is it possible that these dots are fragments of dead cells?

I experienced something similar doing IHC with HEK cells, if you merge DAPI with the bright field many times they are some sorts of nuclear debris.

Dear Mafalda Farelo,

I would propose to adapt the protocoll. For reference see (amongst others and recently published):

Schulz et al., Elife. 2019 Mar 22;8. pii: e42068. doi: 10.7554/eLife.42068.

or

Rottensteiner-Brandl et al., Materials (Basel). 2018 Oct 1;11(10). pii: E1880. doi: 10.3390/ma11101880.

This is mycoplasma infection. You can try mycoplasma removal kit for 2 weeks and check again by DAPI staining or mycoplasma PCR kit

DAPI staining with healthy cell will only present at nucleus but not cytoplasm.

Hi, all.

Everybody who has already dealt with Mycoplasma infection has this fast response against it when these dots are seen. I do feel like you have it but, If it really is, is on its infancy ... Anyway, I would like to tell you to check a few things.

Have you checked the toxic effects of your transfections system and gene? Can you use a marker to check if it is cellular debris? If so, do it ...

Last, you can always try the major treatments for Mycoplasma in your cell line and see If you get rid of these speckles. Ok?

Hope it helps. ;-)

IDK about mycoplasma because the magnification is a little low, but you need to check it out. Since the non-nuclear DAPI positive spots dramatically increase with DNA, might the staining be liposome-DNA complexes adhering to the substrate & cell membranes?

Do you possibly just see remainings of your transfection plasmid? How about changing the culture medium a while before washing and fixing the cells?

As you can see figure [no transfection_dapi.jpg], cell treated with DAPI staining only showed cytoplasm positive. This could tell us that cells are contaminated by mycoplasma.

Transfection leads to cell death and presented small pieces around the cells. This could be the apoptosome which contained cell DNA so that these small particles showed DAPI positive.

You can treat cell with mycoplasma remover kit or thawing a healthy cells and then staining with DAPI preliminary. After confirmation cell are healthy, further transfection experiment will be significant.

FWIW, DAPI staining is a relatively insensitive for mycoplasma detection method, maybe 10EE5 cfu + you need to scan stained cells carefully, as not might be positive. PCR kits can detect between 10-100 cfu, but these kits will not distinguish between dead and live mycoplasma, so not necessarily a good way to tell when treatment has eliminated infection. Mycoplasma enzyme detection methods will detect live critters, so may be your best option. I've included a very positive DAPI myco staining.

Thank you all for your insights.

- Meanwhile I have tested all my cell lines, media, plasmid solutions and transfection reagents for mycoplasma and all came back negative.

- Since I see these specks even outside the cells' cytoplasm, I have also done a mock transfection, where I added transfection reagent and DNA to a glass coverslip (no cells). After DAPI staining I see the glass full of blue dots, but I do not see dots when "transfecting" using only the reagent and no DNA.

- I have tried other reagent (Fugene 6) and I still see specks, and again no specks in non-transfected cells. However, I think these specks are a bit different (see image bellow).

- I have also tried to wash the cells several times before and after fixation and even added one overnight wash with PBS, without success.

I think these dots are liposome-DNA complexes adhering to cell membranes and to the glass coverslip itself. Any ideas on how to get rid of this?

(Image: A549 cells transfected with Fugene 6 with a pEGFP-N3 plasmid and stained with DAPI 24 hours after transfection)

Mafalda Farelo if it is liposome-DNA complex, it should disappear as you culture the cell for longer time. Could it be ecDNA (induced by transfection or expression of the exogenous protein)?

Hi Mafalda

First, the contamination of mycoplasma originates from the experimenter themself, so it is often meaningless to clean the machine and reagents.

Probably, you detected mycoplasma contamination, however, dead cells show strong autofluorescence (you can check "STAP cell"s story). If you can not detect mycoplasma contamination by PCR, autofluorescence may have been detected.

Before doing transfection, you can do cell passage (at least two times).

If cell viability is above 95%, you can start DNA transfection.

Hisashi IIZASA

Hello Mafalda,

Im experiencing the same problem with you. Do you solve it? Thanks!

I have the same exact issue, has anyone found solutions??

https://www.researchgate.net/post/Lipofectamine_Reagents_not_entering_cells_DAPI_small_dots_in_immunofluorescence_contamination?isAnswerFieldFocused=true