It is possible to sort monocytes from fresh PBMCs using CD14 magnetic beads from Miltenyi (or any similar approach) and directly stimulate monocytes with LPS or LBP.

In my experience, sorting monocytes from thawed PBMCs or freeze/thaw sorted monocytes works less well than using fresh cells (because of the difficult-to-predict and difficult-to-measure freezing-related cell alterations). In this case, I would at least monitor cell viability by FACS in parallel for all thawed cryotubes.

Now, whether it is better to use monocytes or differentiated monocytes is really up to you and to the exact question you want to address. All the suggestions you made (using adherent cells or differentiate sorted monocytes with growth factors) are possible. Regarding your question : "Which cells are best to use to get a strong cytokine expression?", my answer would be that it depends on the cytokines you want to measure. For instance, LPS could induce a strong IL-1beta/IL-6/IL-8 production by monocytes but would only induce measurable decent levels of IL-12/IL-23 by monocytes-derived dendritic cells.

Also, bear in mind that in all cases you will also lose some cells as compared to a situation where you directly work on sorted monocytes, so the net amount of cells you have in your cryotubes and the variety of tests you want to perform should be considered for your experimental design.

Finally, there are more and more concerns about cell culture in vitro biases (which are cumulative, i.e. sorting is probably less biased than sorting + differentiation), and it is increasingly considered that working with whole blood could give more reliable information about cell reactivity in a given individual.

Hi Annica

freezing kills the monocytes. Thus, better do the assays directly.

Freezing works much better with lymphocytes, but not with monocytes.

The best way to isolate monocytes is via negative isolation, there are kits for this.

Best regards

Matthias.

Hi Annica,

we have only experience with MoDC from Monocytes (Monocytes isolated with CD14 MicroBeads from Milt.). Important is what kind of LPS you use. We use LPS from Sigma L5014 and it works very well (other partically not). We observe that some MoDC are adherend, most not. We measure the cells separate (FACS our marker CD80, CD86, CD1a, etc.) there are no differenc. So we think a little part of all MoDc are for a period adherent. In opposite to Matthias we have no trouble with frozen cells.

RPMI-Medium: 10% FCS hitzeinaktiviert

4mM L-Glutamin

Penicillin 50 IU/ml

Streptomycin 50 µg/ml

RPMI-Einfriermedium: 30ml RPMI-Medium (s.o.)

10ml DMSO

10ml FCS hitzeinaktiviert

-80C° as far as 3-4 month.

good luck

Jochem

I am very grateful for your good answers! Thank you all three of you! I will try my frozen cells once and then make fresh isolation.

If you are primarily interested in the biochemistry if these molecules then you are better off starting with human macrophage cell lines rather than frozen ex vivo samples.

I used CD14+\CD16- frozen cells and they responded very good to LPS in regard to IL-6, TNFa and more. Thank you for your advises!

Annica