Hi guys! Thank you so much for your notes. Actually, I use the protocol described by Lutz etl., 1999, and after the differentiation I take only the loosely adhered cells to my experiments. But when I plate those loosely adhered cells, they attached really fast to the 96well bottom plates, and that is when I have the dettaching problem. I will try all your suggestions and as soon as I find out what is better I will let you know. Thank you!

To my knowledge DCs are not suppose to adhere that strong to plates. I used to differentiate the bone marrow cells in coated dishes and for harvesting the DCs I only rinsed the bottom of the plate (~10 times) with medium using a pipettor. Only the loosely attached cells will come of, this suspension contains a lot of DCs. The cell layer that stays behind is partially made up of macrophage like cells that are known to adhere well. Replating the DCs should be done in non-treated plastic dishes as the DCs could be activated by the coating that is often used in plastics for tissue culture. Detaching the cells should be relatively easily with PBS/2 mM EDTA, 5 minutes at room temperature or at 4 ºC (then it takes a bit longer for the cells to detach). So, harsh mechanical treatment of the cells should not be necessary. Alternatively you might try to do your experiment using cell suspension instead of plated. Hope this helps......

Try with ice chilled PBS, add 2ml in a flask and keep it for 5-7 min at RT or in incubator.

If you still find some of cells remain attached; try with 0.5mM EDTA in PBS instead of PBS only and use it chilled

T25 flask- Treat cells with 2ml of chilled buffer for ~5 min at RT/incubator

Add 3ml further and rinse with pipette

Wash and take the pellet

I hope, it will help

Best

Hi Ana!

As Toine commented, the type of plastic that you use is very important. The easiest solution is to use in your experiments plates for suspension culture (instead of plates for tissue culture).

If it is not possible for you to purchase these kind of plates, I suggest you the following test: count your cells and split your them (without any drug treatment) into 6 groups: (1) chilled PBS with 2 mM EDTA+10 min incubation at RT or (2) 10 min incubation at 4ºC;  (3) chilled PBS with 0.5 mM EDTA+10 min incubation at RT or (4) 10 min incubation at 4ºC; (5) chilled PBS (no EDTA)+10 min incubation at RT or (6) 10 min incubation at 4ºC. Gently wash, recover the cells, count them, and analyze their viability by flow cytometry with your standard protocol. Then you can analyze which treatment gives you the highest % of recovered cells with the highest % of viability. 

Hi Ana!! Me I add to my adherent cells cold PBS and incubate them at 4°C during 20 min and it works wonderfully.

Best

Dear Ana, good morning

I agree with the previous comments that DCs usually do not adhere strongly to culture plates. Are you using only GM-CSF for cell culture? It is important to induce differentiation of DC to add GM-CSF plus IL-4. In this condition you can differentiate approximately 50-70% of bone marrow HSC to DCs. The remainder cells are differentiated to macrophages, that adhere firmly to the plates. In this condition DCs detach easily after treating with chilled saline (or serum free culture medium).

Hope it helps.

All the best

Please try cell dissociation buffer from Gibco; it comes without trypsin. You need to remove serum before detachment; afterwards, just pellet and reconstitute in medium since this neutralizes the buffer.  We found more than 85% viability.

use a protein free ca free solution at 4º and a rubber policeman to scrap them.

Well, to my knowledge, those cells strongly adhering to the bottom are likely to be macrophages (also when looking at the morphology), see original protocol by Lutz etl., 1999, J. Immunol Meth), lthough, there is, of course, some plasticity...

Otherwise, for bovine DC we also used cell dissociation buffer (see comment from Dr. Jagannath), worked fine.

Hi guys! Thank you so much for your notes. Actually, I use the protocol described by Lutz etl., 1999, and after the differentiation I take only the loosely adhered cells to my experiments. But when I plate those loosely adhered cells, they attached really fast to the 96well bottom plates, and that is when I have the dettaching problem. I will try all your suggestions and as soon as I find out what is better I will let you know. Thank you!

Hi everybody!

I agree with all your advices: remove serum from the media, macrophages very attached to the plate....

Some years ago I used non-adherent plates and it was ok, but as well as my work with conventional plates was succesful I never used it anymore.

My "big" advace, be very very careful with the system that you use to dettach the cells because can find yours DCs in different differentation states!

My experience is with porcine and bovine monocyte derived DCs.

Hope something from my reply be useful for you.

Hi Ana,

Agreed with Dr Jagannath advise. Otherwise, you may use the same medium without serum but make sure it is cold. Incubate for about 5-10 mins and flush all the cells that attached to the surface and process faster to avoid more dead cell.

we do not use adhesion plates to perform such experiments...with suspension plates, 2mM EDTA for 2 min is sufficient 

Hi Ana, according to your second reply,

when you transfer those loosely adhere DC to 96 well plate, try to use rmGM-CSF of lower density as comparing to your first day dose. then you try your experiment with your drug for testing with overnight incubation. 

Yeah

I have the similar problem

DCs are tightly co-cultured with lots of

In my experience if you use un-treated plates (like plastic petri dishes for bacteria culture) and you add b-MeEtOH (1:1000) to the culture you can recover the cells really easy using only chillled PBS