Who uses non-adherent cells? - like NCI-H69... are they ready to use after passing? (Jul/09/2008 )

Hey there!

I'm new in working with non-adherent cells, NCI-H69 to be precise. In an adherent cell line, I usually do the experiment 12 hours after passing cells, mainly to let the cells attach to sustrate and divide and so on. What would you do in case of using non-adherent cells? They don't need tripsynization, just a wash and media change. I want to stimulate with a drug cytoplasm-acting. Would they be ready to use after passing? How many time would you wait to start experiment after passing?

Thanks for your advices.

Aleruiz

Hi Aleruiz,

With regard to working with Suspension cells, it's as straight forward as you think... Wash and Use.

Simply centrifuge them, wash once, and then resuspend in whatever buffer you'll be working with.

I'll give an example. Let's say i'm going to do a FACS analysis on CD32A expressed on K562 cells.

K562 cells are a WBC line and are a good model for monocytes, neutraphills, and immature macrophages... They are a suspension cell line, and like your NCI-H69 cells they tend to grow in clusters but not completely.

Facs Buffer - RPMI/ 1% ultra-low IgG FBS / EDTA 3 mM / NaN3 (0.05%)

1: Centrifuge cells
2: Resuspend in colf facs buffer
3: Cell Count
4: Centrifuge cells
5: Resuspend in cold facs buffer at a cell concentration of 10 million cells / ml
6: Add primary antibody
......................................

There's more steps after that but you can see from the first few steps that there is no trypsinization or what not...

WARNING!!!!
I noticed you wanted to stimulate the cells... Even when working with Adhesion cells, if you are investigating some sort of cell surface receptor (which I suspect you are) trypsin is likely to cleave your receptor... You NEVER want to use trypsin on cells before you run an experiment involving their surface receptors... using trypsin on them the day before, in most cases, should be fine as they have time to re-express the surface receptors...

QUESTION!!!

I'm curious why you are using NCI-H69 cells... I'm also very curious as to why they are suspension cells... I took a look in the ATCC database and NCI-H69 cells are a human lung carcinoma cell (i'm not sure if they're epithelial or endothelial) but in either case I find it odd that they are suspension cells. Mind you, I'm NOT saying they AREN'T suspension cells, I'm saying I don't understand why they ARE... Do you know?

Also, I'm wondering what you're trying to model. NCI-H69 cells have an odd number of chromosomes, so are you modeling some sort of condition which involves having an odd number of chromosomes?

Just so you know, I'm only asking because I'm CURIOUS...In no way am I saying you're doing anything wrong...