Hi all,
I am currently struggling to obtain a high number of immature DC from bone marrow progenitors.
For example, from 4 tibias and 4 femurs (Balb/c female) I can have around 55x10^6 progenitors, with a yield of immature DCs of about 4-8%, which is clearly to low.
I am aware that there are many variables to consider.
Briefly, my protocol is:
- 5x10^5 progenitor cells in 2ml (complete RPMI1640+10%FBS) seeded just in the middle of 6-well plates + 20ng/ml rmGM-CSF
- day3: addition of fresh medium + rmGM-CSF
- day6: removal of 2ml old medium and addition of 2ml fresh medium + rmGM-CSF
- day9: harvesting immature DCs and plating at concentration of 1x10^6 cells/ml in 24-well plates (in 1ml) + 1ug/ml LPS +/- desired stimuli
- day10: study of mature DCs.
Main differences with other protocols I have found so far:
- non-TC grade petri dishes (from day0 till immature DCs) = tried (2x10^6 cells/dish) but the result was the same.
- red blood cells lysis=I do not do it.
- fungizone (amphotericin B)=present in my medium.
- scraper=I use it, some don't.
- feeding every 2 days (day2 and day4) then getting iDCs on day6 and mDCs on day7=tried, but same results.
At microscope they look very good, but the number doesn't seem to be good.
Please, help! I have been loosing time and money since December...
Many thanks to all of you who will be replying!
FC
Hi Francesco, around 5x10^7 progenitors is a common result from one Balb/c female, so the harvest of the cells seems to be o.k. However I would recommend a RBC lysis since RBCs consume a lot of nutrients from the medium. But overall we use a similar protocol with much better results (90-95% of CD11c and MHCII positive cells). The only differences that I find is that we do the complete culture procedure in 24well plates with only 5% FCS instead of 10% and a cell density of 1x10^6 cells. The GMCSF that we use comes from Immunotools it is cheap and works well but we use it at the same concentration like you. And we do not use Amphotericin (never had any contamination with fungi)! Thus I would recommend to omit the Amphotericin and lyse the RBC by hypotonic shock. Good luck!
Hi Francesco,
You can also try density gradient to remove unwanted cells like RBCs.
I did not get whether after the culture your over all cell yield is low or proportion of DCs in culture is low. But it may be the issue with the GMCSF. You can try rH-GMCSF from peprotech you can also add IL-4 too in the culture (5-10 ng/ml). Sometimes stimulation do affect DC viability. It would be nice to analyze the cells before stimulations.
Best wishes.
change the site of collection, the preferred site is the rear upper pelvic bone (posterior iliac crest), since no major blood vessels or organs are located nearby.
A second location is the front upper pelvic bone (anterior iliac crest).
hope you doing the best.
Dear Marcus,
Thank you very much for replying. Your response was very helpful. I will be trying to introduce the modifications you suggested as soon as I can.
As a matter of fact I also performed the FACS analysis after I wrote the question. Around 90% of cells were CD11c+ and MHC-II+ as you said.
But I have to say sorry for not being clear about the yield. With the term yield I wanted to mean the number of DCs obtained from progenitors: i.e. if I have 55x10^6 cells from the bone marrow in total, I will be able to have around 4,4x10^6 differentiated iDCs in total. :'(
Could it be the scraping another main reason for the massive death of my cells?
Thanks again!
Dear Hemant,
Thank you for replying and for your suggestions.
Unfortunately, I cannot use IL-4 because I will be needing to assess the production of that cytokine as part of the experiments I have to do.
Dear Mosab,
Thank you very much for replying. I will take that information into account for getting a bigger amount of cells.
Dear Francesco,
around 4x10^6 are indeed a low yield. I would also suspect the cell scraper... we never used it for harvesting BMDCs. We usually harvest them by repeated flushing with ice cold PBS without Ca and Mg. Its a littlebit laborious but gives us the best yield and vitality of the cells.
Good luck!
Dear Marcus,
Perfect. That's another important piece of the puzzle. I'll definitely do the way you said.
Many thanks again! Very helpful!
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