Does anyone know how to keep GBM cell lines (U373, SW1783, LN-405, DBTRG and U87) in suspension and viable for the duration of FACS? I have tried several things including adding EDTA, DNase, using Accutase and Accumax, keeping on ice and so on. Viability isn't so much of an issue but they just seem to stick together a lot, resulting in a low percentage of singlets. Any advice is appreciated.
Thank you
Dear Melanie Maerken ,
would you mind sharing a little bit more about your protocol? I used to perform flow cytometry staining on U-251 MG, U-373 MG, and T98 G glioblastoma cells, and I know how frustrating performing flow cytometry in these cells can be.
The primary concern was that all the glioblastoma cells (including several of the ones you mentioned) are very big in size and difficult for you to achieve a high number of events at all. Additionally, they slip very easily from some kinds of plasticware that we may use for staining. I normally used eppendorf tubes (1.5 mL) for my flow cytometry protocols, but for glioblastoma cells this was impossible, as for each centrifugation and washing steps I kept losing more and more cells.
The best tubes you can use are the cytometry tubes (5 mL) and never remove fluid by inversion. You should always start with many (many) cells, perform very gentle centrifugation (because they precipitate rather easily) and keep them on ice when you can.
I only detach cells with a very low concentration of EDTA (20 min with gentle agitation), and never had a problem with it. During staining, I kept cells under gentle agitation as well. Be sure to give a flick to the tubes once in a while, because they do precipitate quite often.
If there is anything else I can help, please just ask.
Thank you.
Hi Eduardo Listik
Thank you very much for your response.
A few more details about my protocol. I harvest the cells using either 0.05% Trypsin or Accutase, centrifuge the cells at 800rpm for 4min and resuspend in PBS and Accumax (1:1) to count the cells. I then perform further dilution to have a final cell concentration of around 3-5 million cells per ml. As a sorting buffer I just use PBS and Accumax but I have tried also other buffers such as PBS+1%FBS+ 5mM EDTA. My main problem is that these cells aggregate and sink to the bottom or they adhere to the stream line. In general we have issues with clocking and not getting a steady flow and overall we have a low percentage of singlets which means I loose over 50% of my cells during the sort due to them not staying in suspension.
Could you please share with me how much EDTA you use to detach the cells and also what buffer you have used to sort the cells?
My cells express fluorescent proteins so I don't perform any staining.
Thank you
Melanie
Dear Melanie Maerken ,
are you filtering the cells before FACS? This normally remove the clumps formed when these cells precipitate (as they do very often).
To detach the cells, I use 300 uM of EDTA (just leave 20 min, and give some flicks every 5 min, you can remove cells easily with a pipette). In the buffer you can increase EDTA ten fold (3 mM). You should always have in the buffer (1–2%) FBS and (10–25 mM) HEPES as well. I normally used 1% FBS, 25 mM HEPES, pH 7.0.
Hi Eduardo Listik
Yes, I do filter the cells before FACS and even vortex the cells, resuspend and filter again during the FACS.
I actually use higher EDTA concentrations than you mentioned in my FACS buffer but unfortunately it doesn't improve anything.
I will see if detaching the cells with EDTA instead of Trypsind will help. I assume you dilute EDTA in PBS?
Thank you