12# and 6# is ok. I used to culture in 6# and treat with PHA. The cells concentration should been same.

How much volume, how much cytokine...

Thank you! I would like to have around 1-2 ml of supernatant for further analysis. Plus number of patients for the study does not usually excede 2. So I afraid that if I use 24-well plate, the majority of it will be left unused and waisted.

Hello Hyde Chen ,

Could you tell me the concentration of PBMC in 6-well plate in your protocol? Because now I also culture PBMC with or without PHA in the 6-well plate for some days. But it is very strange that I always get the low cell viability in FACS (about 55%) after culture.

I cultured PBMC at a density of 1x10^6 cells/well in 6-well plate in 2ml complete RPMI medium. I found someone reported that the low concentration of PBMC might affect cells survival.

So I think maybe this result from the concentration of PBMC?

Thank you so much!

Hi every body, PBMC+PHA can cultured well in any format of Plate but you should have high concentration of PBMC and more than you think. for example for 12 well 1x10^6 cells and for 96 well 250000 cells. they feel better when they are at high dencity.

Bahareh Khalaj Hello Khalaj,

Have you tried to perform PBMC culture in 6-well plate? Could you tell me what the concentration of PBMC you uses? I dont know why I always got low cell viability of PBMC after culture in the 6-well plate for 2d without PHA.

Thx!

Hi Junfen,

PBMC without PHA or IL2 or any other stimulation factor can not grow and proliferated. you will loose cells which means low viability. but with bra medium you can decrease the loosing percent. use Advanced RPMI+10%FBS+2 mM glutamin+ penicilin. I dont worked much with 6well but 2X10^6 should be worked.

Hi Khalaj,

Thank you for your response. Because I need the PBMC alone as my control. I found most people would like to use 24-well plate to culture PBMC at a density of over 1x10^6 cells/ml.

I also want to know that before you culture PBMC in the plate, is it necessary to assure the cell viability of PBMC is over 90% or not? Normally I use frozen PBMC for my experiment from the nitrogen. But after thawing, the cell viability is low, about 70%. So I think this is also one of reason to explain the low cell viability after cell recover from the plate.

Junfen

Hi Junfen,

So you probably consider PBMCs without simulation as your negative control. As Bahareh mentioned, PBMCs culture without stimulation results in no growth and less viability.

About your next question, we usually use fresh PBMCs and check the viability after isolation through a viability die such as PI (by flow cytometry) or trypan blue. Also, it is natural that you see less viability after PBMCs thawing. For getting better result you need to freez PBMCs in a higher count rather other cells about 20-30 million cells in freezing media including 90% FBS and 10% DMSO. Then it is better to transfer it to -20 for hours, -70 for days and then to nitrogen for months. It should help you to get better result. After thawing, you will see dead cells. Give them time (about 3-4 hours) to optimize and recover. Then refresh the media to remove dead cells and debris and continue to your desire culture. Hope it helps.

Alaleh

Hello Alaleh,

Thank you for your response.

I would like to know how to remove dead cells and debris after PBMC thawing? Normally I prepared 10ml of warm complete RPMI medium in 15ml falcon tube in advance, and thaw PBMC in water incubator until remaining little ice, then transfer them to the tube drop by drop. The last step is to do centrifugation at 1800rpm for 8min and discard the supernatants to recover cells. But I do not know how to remove the dead the cells? If possible, could you tell me?

Thank you so much!

Junfen

Hi Junfen,

as I know, you can not seperate dead cells after thawing but you can consider your cell count on live cell concentration after staining with trypan blue or PI. another thing you should know that most of cells which are died after thawing are granaulocytes which can not proliferate more. another things is that when you thaw your PBMC, dont move them at water bath and keep them stable on the water bath. another thing is that you can keep PBMC in medium for overnight after thawing and then centrifuge them and change medium as Alaleh mentioned. I hope all these tips help you. Best regards

Bahar

Hi Bahareh,

Thank you for your tips! They are very useful! I will pay attention to these in my future experiments!

Junfen

Hi Junfen,

You can remove dead cells by Centrifuging them (at 5 minutes 200g) as Bahareh mentioned. Please note that the centrifuge should be performed after the cells are incubated for 3-4 hours in medium post-thawing. The point is that the more volume you centrifuge the cells, the better result you will get. As a researcher pointed out, "Re-spinning of the supernatant at 1500 g for 5 min will allow you to see a thin pellet represented by dead cells and confirm the separation" .

Good luck!

Alaleh

Hi Alaleh,

Thank you for your advice.

So you meant that after centrifugation at 200g for 5min, dead cells are the thin pellet in the bottom of the tube?

I think you wrote the 1500g by accident? As 1500g is too high. Is it 1500rpm?

Junfen

Hi Jufen,

200g 5 min is for removing dead cells (medium on top) from your alive cells (pellet in down).

1500g (for the medium containing dead cells) is just for further confirmation that assures you about removing dead cells.

Alaleh

Hi Alaleh,

Thank you so much for your patience!

Junfen