In my opinion, you can try to leave the sample in incubator no more than 30mins, because much more time it remain in incubatore, much more contamination (B-cells and residual platelets and red blood cells) you will collect.

Also, I think that it's better (if you can!) work with freshly samples.

Good Luck!

Hi Aesha,

I have some Question. First: you get the frozen cells in cryomedium - the cryomedium contains ? Second: there are all kind of leukos in the cryomedium, but you need only monocytes - right? Third: how do you intend to separate the monos ?

I think you have to remove as quick as possible the cryomedium after thawing.Either you separate e.g. with magnetobeads directly or thaw them and separate later with an other technique. It is very helpful to describe what you do more detailed and say what the aim. You can use frozen cells but of course it is better to use freshly samples. In our Lab we use freshly samples but after separate the Monocytes we freeze them and store until 3 month - it works very well. Our cryomedium contains medium RPMI 1640 (w/o Phenolrot) 15% FCS and 10% DMSO.

All the best

Jochem

It is not recommended to cryopreserve monocytes at all. They have poor viability and the once that survive do not resemble responses from fresh monocytes. You will have to us fresh cells.

After isolating your PBMC you can enrich for monocytes by MACS isolation using eg CD14 beads or use an adherence procedure. The adherence procedure is cheaper of course, but also will give lower purities. However, when you use a differentiation medium, eg GM-CSF/IL-4, your purity will generally improve during the days of differentiation.

Hi Aesha,

maybe its helpful

Jochem

Hi Jochem

First: you get the frozen cells in cryomedium - the cryomedium contains ? Second: there are all kind of leukos in the cryomedium, but you need only monocytes - right? Third: how do you intend to separate the monos ?

1-My freezing medium is ( 90% FCS and 10% DMSO)

2- Yes Right

3- I let the cells after thawing in the incubator to let the monocytes to stick to the surface of the flask then I wash non adherent cells 3x with PBS ,,, Then, I incubate it with IL-4 and GMCSF ,,,,

This protocol is good with fresh samples as I tried with buffycoats ,,, but because I need a specific blood type (which is cryopreserved ) ,, that's why I need a good protocol for frozen samples,,,

I need dendritic cells to pulse them with virus then use them to check the proliferation of T cells. So if you have any idea about the best protocol to pulse them with virus --> that will be great

Many thanks

Thaanks Gerco

As I said to Jochem: This protocol is good with fresh samples as I tried with buffycoats ,,, but because I need a specific blood type (which is cryopreserved ) ,, that's why I need a good protocol for frozen samples,,,

I need dendritic cells to pulse them with virus then use them to check the proliferation of T cells. So if you have any idea about the best protocol to pulse them with virus --> that will be great

I have no experience with it, but in that case I would look into what people have done using CD34+ progenitor cells. Not sure if you can get enough cells that way, but possibly they are more resistant to cryopreservation?

Hi Aesha,

i have another question. Do you receive the cryopreserved leukopheresis from another place like hospital ? I think you have directly after thawing remove the cryomedium. I would prefer - first separate the monocytes - or is there a special reason to incubate with all the cells ? I have no experience with seperate the monocytes with non-adherent technique, we use magnetobeads (MACS CD14). May be you have to check different flasks. We observe that over the time monocytes a part of them are adherent, we tested both the adherent and non adherend with CD1a, CD14,CD86, CD80 and HLA-DR with the same result and we suppose that the monocytes temporary are adherent. May be you lose with your non-adherent technique monocytes, but this is only a guess. Avoid dead cells in the flask, what you do when you wash the thawed cells.

Jochem