Hello, I am an undergraduate student doing my dissertation, where I isolate PBMCs from leucocyte cones in order to plate monocytes and allow for macrophage differentiation.
Although the numbers look alright on the first and second medium change, towards the end of the incubation period, they suddenly drop and the number ends up being very low.
I use RPMI 1640, Human Serum and GM-CSF. I have used both 10cm petri dishes, 6cm dishes and 96 well plates and the problem remains.
Apart from checking the incubator for temperature and CO2 levels, checking for pH in medium and checking for mycoplasma would you have any other suggestions ?
Thank you in advance,
Ioanna M.
I suggest to use Dutch modified RPMI (Gibco #22409-015) and tested endotoxin-free human serum for MF culture. Corning Primaria plates or flasks support better MF adhesion.
Hello, Ioanna!
Please You look at this article:
Article A protocol for rapid monocyte isolation and generation of si...
For differentiation of PBMC into macrophages, it is necessary not only GM-CSF, but also IL-4. Сheck also your concentration of GM-CSF.
2.3. Complete Culture Media (CCM) evaluation in differentiation of moDCs
After purification, cell density was adjusted to 0.5 × 106 cells/mL. Cells were cultured in 24-well tissue culture plates (Falcon Inc., USA), resuspended in either RPMI-1640 or DMEM culture medium (Sigma Immunochemicals, USA) supplemented with either 10% FBS or 10% HS. moDC differentiation medium (CCM) was obtained by supplementing each culture media described above with 300 or 500 UI/mL of recombinant human (rh) GM-CSF and 300 or 500 UI/mL of rhIL-4 (PeproTech Inc., USA). After 48 hours, half of the total volume of each well was carefully removed, and the respective CCM was added for an additional 2 days.
What procedure are you following for monocyte isolation? Is it Ficol concentration gradient? Read my publication and PhD thesis- It works very well -
Details: Monocyte derived macrophages (MDM) MDM were prepared from healthy donor buffy coats supplied by South African Blood Transfusion Services. Peripheral blood mononuclear cells (PBMC) were separated by centrifugation over Ficol-paque Plus (Pharmacia, Uppsala, Sweden). Cells were washed in RPMI, pooled and counted. Cells were incubated in large tissue culture flask (175 cm2 ) at 3.00E+09 PBMC per 25 ml RPMI per flask for 2 hours at 37degree C in 5% CO2. This step allows monocytes to adhere to the flask. Non-adherent cells were removed by three washes with 10 ml of pre-warmed RPMI medium (Sigma-Aldrich, Ayrshire, UK). Finally, 10 ml of ice-cold phosphate buffered saline (PBS) was added and the flask incubated at 4 deg C for 20 minutes. Using a long handled scraper, monocytes were dislodged from the bottom of the flask and pooled in a 50 ml falcon tube for counting. Cells were plated in R10 medium (RPMI containing 10% fetal calf serum (FCS) at a concentration of 2.00E+05 cells/well in a 96 well tissue culture plate and cultured at 37 deg C, in 5% CO2, for 6 days in R10 medium to mature into macrophages.
You can also try Percol gradient (PMID: 12957415). Column based MACS are costly but best.
I am an academic and scientist with PhD and postdoctorate and all my research has been conducted with utmost ethical approval and ethical considerations.
I need to write this as I was getting lots of unusual interference whilst I was typing this comment via script and it's absurd. It's attacking even my family.
I do not know the remedy for these people. I love to comment and share on my experience and expertise to students, peers and scientific communities.
This is for all scientific community to convey what I am going through since few years! This is a human rights violations going on against me since years!
Sorry Ioanna this is not for you!
I have to write it as I came across of all withstanding capacity of harassments in my digital life!
The question of how you are adherent isolating the monocytes is relevant and information on your technique might provide insightful. Note, human monocytes initially adhere and then some float off of the plastic surface. Typically this occurs 3-4+ hours/ Thus, the timing for the washes at 1-2 hours is critical. Further, re-feeds should add-back the non-adherent cells. You might also do better using M-CSF for macrophage differentiation and maturation. It is possible that the monocytes are undergoing apoptosis due to a lack of the right growth factor. I will note that viability with the use of rubber policeman is poor. Better to use cold calcium magnesium free saline solution with EDTA to remove any adherent cells.
Yes James, adherent step is little critical but we have assessed CD11b and CD14 and HLA-DR expression in this time point and found that 2 hour was optimized for adherent. This is repeated in day 6 as well.
I am little concern about stimulation with M-CSF as it will promote generally M-2 macrophages whilst GMCSF promote M-1 inflammatory phenotype ( PMID: 28637652).
So we have kept it as it is to mimic the natural phenotypes as our objective was for tuberculosis infection study instead of alveolar macrophages.
Thank you very much for your answers.
I use Histopaque for PBMC isolation and EDTA for lifting.
I also use human serum, RPMI and GM-CSF as I mentioned above. Do you think I should use a different kind of serum (i.e. supplemented with Calcium ) ?
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