Hi All, I am using cell mask orange dye to label HEK 293 T cells plasma membrane. I am using 5 ug of dye as final concentration and incubating cells for 10 min at 37•C. I am facing issue with dye internalisation. I did staining before fixation as well as after fixation. In both case All dye internalize. So here is the protocol I am using:
1. Growing cells
2. wash with PBS
3. Stained with cell mask orange for 10 min
4. washing 3 time with PBS
5. Fixation with 4% PFA
6. Washing with PBS.
7. Stored in PBS,Same day Imaging.
Note: Some time I pellet down the cells after second step and then stained the cell suspension. At the end step I just mount the sample on slide.
How can I prevent dye internalization in cell? what are the possible reason of this issue? Please let me know.
I would try labelling for a shorter time period - I find sufficient labelling with this probe in about 5 minutes at 37
What is the concentration of the dye 5ug in what volume?
Edward Sanders I used 1.8 mL 5 ug for 35 mm dish. Thank you for the answer. Should I decrease the concentration too?? with the time?
I'm with Thermo Fisher Scientific tech support.
There are a number of potential issues to consider, more than can be discussed easily here. But we first need more information from you.
Please write us at [email protected]. Please include the catalog number, lot number, storage conditions of the stock, the staining concentration (not mass amount), and your protocol. Though I am the supervisor of the group, please feel free to address the email to me if you wish.
Jason A Kilgore I did that already last week. They suggest me to play with Incubation time and concentration.
Shoyab Ansari
The 5ug doesn't make that much sense?
Isn't cell mask usually supplied in 1000X stocks?
So to make a 1 X stock you would need to make a 1 in 1000 dilution from the stuff you bought.
@Edward, Concn of Dye is 5 mg, I did dilution accordingly. Likewise you are suggesting.
Shoyab Ansari I found your email thread with our Tech Support. I am replying to it with some additional thoughts. Please feel free to reply back if you have any questions.
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