Hello everyone,

I'm using CellTrace Far-red proliferation assay (Thermofisher C34572) for the first time,

I'm doing it on human embryonic stem cells at a time and for 2D differentiated cultures coming from the same type of cells , so here is how I do it :

A- For stem cells: I use adherent cells protocol as follows :

- Cells are in a 12 well plate.

- wash the cells with PBS

- add 300 µm of staining sollution (PBS+ 1 µM CellTrace FarRed)

- incubate for 20 min at 37C°.

- take out the staining sollution and add 500 µl of DMEM +10%KOSR, incubate for 5 minutes at 37C.

- replace with mTesR plus medium and incubate for 5 days ( in hESC, the generation time is about 20 hours).

B- For 2D differentiated cells:

I use an otic fate differentiation with media and timing similar to this protocol (https://www.cell.com/cell-stem-cell/pdf/S1934-5909(23)00217-5.pdf) but in 2D, I differentiate for 9 days, start the staining, and incubate for 5 days then analyse.

for these cells, I use the suspension staining method:

- dissociate cells into a single cell solution with accutase.

- spin down 300 000 cells.

- incubate with 300 µm of PBS+1µM CellTrace FarRed for 20 minutes with shaking.

- add 5 times volume of DMEM +10%KOSR, incubate for 5 minutes.

- spin down, resuspend in differentiation medium and incubate for 5 days, then analyse.

The problem:

the results seem a bit skewy, the pics seem to be fused and not seperate, even though the cells have clearely divided and the wells have become confluent.

I'm also wondering if for the generation 0 pic, if I should do the staining for 1 day for each cell line to figure it out.

see the attached images for the anlysis pics.

Any help is highly appreciated, thanks in advance !