Hi Agnieszka,

Try two changes. Use Ficoll-Hypaque density centrifugation and use 10% human serum. Good luck!! Use Pen-Strep antibiotics.

Dear Debanjan

Thanks for the answer, but the isolation is not the problem - the culture is.

And there are almost no dead cells, so I don't think the media is cousing it...

Thanks anyway!

I understand the problem. Ensure that you dilute the whole blood initially and use the Ficoll at warm temperature (very important!!) Use 12 ml Ficoll column at 20-23 degree celsius. Use a 50 ml centrifuge tube.

Buffy coat dilution: 1:2 with PBS (without Ca/Mg) at room temperature!) 

Hi Agnieszka,

you gonna lose the monocytes (up to 20% of WBCs) due to their plastik adherence. So you can vigorously wash by pipetting. I usually did that washing step 3 times.

Another option is using cell scrapers to harvest the cells. You gonna harvest also a proportion of that adhered monocytes. You will end up with some debris of the dead cells so it might be usefully to spin them with a FCS gradient.

Good luck

Hello Ingo

Thanks for the answer.

Do you think that using non-treated plates and cell culture tubes (like those: http://www.tissue-cell-culture.com/docs/TC/tissue-culture-tubes.html) could solve the problem?

Hi Agnieszka,

If you do a CD14 staining of your samples you probably will find almost no CD14+ cells by flow cytometry, that will explain at least some our cell loss.

I'm not sure that the equipment of the link you posted will help. Generally people use this property of monocytes’ plastic adherence as a cheap, quick and easy way to separate monocytes from lymphocytes (e.g for differentiation purposes like generation of dendritic cells or makrophages). So the “problem” is more the plastic surface rather than the shape of the container you’re using. I know that some people use Teflon coated bags to generate macrophages trying to minimize adherence and ending up with a higher yield.

To me the more relevant question is: what are the cells of interest for your analysis? If it is lymphocytes you’re fine the way it is… if the problem is only cell numbers try using buffy coats e.g. so you should have plenty of cells for any kind of subsequent analysis.

You probably can get buffy coats or the like from the guys of your hematology department or at least they can help out with inform where and how to get those.

Hope that was of help

Ingo

Hello Ingo

I'm trying to analyze expression level of urocortin 2 in PBMC. I don't really care what type there are. My problem is that in PBMC there are small amounts of RNA - we obtain around 0,6 ug from 10^6 cells, but when there is less than 1mln the amounts get smaller and final concentrations are very low... To small for efficient RNA amplification for microarrays (Illumina)

Hello Agnieszka,

so your main problem is quantity. If possible use more blood, 10 mL seems quite a small amout to me, or as I mentioned above buffy coats (that should be PBMCs from roughly 500 mLs blood donation...so plenty of cells to work with)

Unfortunately I can't do this, I need to use the blood from a single donor...

But thanks anyway for all your help and suggestions

Agnieszka

you are welcome!

buffy coat is from single donor... or do you mean a certain person

1 patient (mostly a sick one) = 1 blood sample = 1 culture