I am new to fluorescence microscopy and i am doing a transwell migration assay for two cell lines (HepG2 and SNU449). I am still optimizing the technique with non-treated cells. My procedure is as follow;
1-seed 150,000 : 200,000 cells in a300 ul media contains 2% FBS in the insert, while adding 600ul of media with 10% FBS in the lower chamber.
2- incubate cells for 18 h
3- wash inserts twice with PBS, then fix with 4% formaldehyde+ 1% triton-X100 for 15 min , and wash twice with PBS
4- stain with DAPI (1:10000) for 15 min, then wash twice, and take photos on a fluorescent microscope. ( i had to repeat this step twice as i first visualized the inserts with an old microscope which gave a very faint blue color)
Now; I can find that in a lower magnification, there are many bright blue dots (other than the rounded nuclei), and those dots are either inside the nucleus itself or outside. Also, there are many black dots inside the nucleus which i assume they are the pores of the transwell insert itself. (does that mean i should leave the cell for a longer time? ) taking in account that after incubation time (18 h) i found the color of the media in the lower chamber has changed which might be a result of cells migrated and fall into the 24-well plate itself or maybe cells were still attached at the bottom of the inserts but utilizing media in the lower chamber?
So my questions;
1- what are the bright dots? and blue patches in my photos? are they apoptotic bodies or a cluster of fragmented nuclei? or it is just the microscope focus; i wasn't able to take good photos?
2- in photos (SNU1 & SNU2), there are stretches or filamentous shapes as if the cytoplasm was also stained ? does anyone have an idea what could that be?
2-since i have the two events happening at the same time; 1- a change in color in the lower chamber (suggesting higher density or higher incubation periods) and 2- cells trapped in the inserts pores= the black dots ( suggesting short incubation time), how can i solve that ?
The bright spots are areas of heterochromatin, chromatin foci, and are not a cause for worry.
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