I'm trying to quantitate uptake of a fluorescently labeled polymer. After incubation for 8 hours for different concentration, I trypsinize the cells, spin them down. Resuspended them in dpbs with 10mM EDTA (since HepG2 cells grow in clumps) down again and fix them in 2% PFA for 30 minutes on ice.
When I perform the flow, the dot plot has 3 different regions (although not distinctly different). The subpopulation which is the largest is autofkuorescing whereas the rest aren't. I'm not sure what I'm looking at and why the HepG2 cells are so diverse.
Any help would be greatly appreciate! :)
Dear Usama Abbasi,
I will suggest to run a parallel unstained control with no incubation of probe and consider it your background control. Take care that you don't have too much of cells in minimum volume as that may be another factor for clumping. HepG2 is a cancerous cell line so yes, you can observe a heterogenous population of cells and resulting different clusters (as they are not so distinct so there is a heterogeneous population). You can try minimizing clumping by doing so. Try to handle the cells very gently. You can use a p1000 pipette for this. Use these unlabeled cells to gate the non fluorescent population and set your gate at the end of peak. Your autofluorescence issue will be resolved by doing so. Now the cells which will be coming beyond peak in probe incubated samples will truly represent the population labelled with probe and intensity profile you can easily obtain as MFI.
Hope it helps !
Hi Usama, I think you see cell agreggates if the population seems to be the same but higher, like a duplicate of that population. Try to resuspend it better (but gently) by using a p200 pipette, to avoid the clumps.
Do you use 2% FBS in your PBS buffer for wash after stainning? It will avoid the unspecific binding of your antibodies :)
Hi:
little tips for your reference:
1.Use FSC-A vs FSC-H dot plot to eliminate doublets
2. 5mM EDTA
3. Do you really need to fix your cells? If not, run samples on flow cytometer immediately after preparation.
4. you may need live/dead reagent (impermeabile dyes) to remove dead cells from data.
best wishes.
Is it possible to replace your HepG2 with another HCC (e.g. Hep3B)? It's one of the worst cell to study in flow cytometry works becuase of clump formation. If it's not possible, I also recomend the same things above. Additionaly, working with high confluent HepG2 cells (>80-90) or seeding HepG2 (lower than 30-40%, after division cells cannot be separated well compared to higher conflunt cell population) lead to cells forming clumps easily.
Thank you everyone for the valuable input! I made some changes to the protocol and now it's working out well. I've opted to not fix the cells (adds a considerable amount of autofluorescence) and to use an live/dead stain.
Also, pipetting the pellet up and down seemed to do the trick. I appreciate all the help with this! Thank you :)
- Badges
- Science method