Hi Cemre Kirşan

The detachment and death of monocytes during differentiation to macrophages can be due to several factors. Ensure your plates are tissue culture-treated to promote cell adherence. The absence of serum during the initial 2-hour incubation might cause cell stress; consider adding autologous serum or a low concentration of FBS during this step. Verify the quality and concentration of M-CSF, as it's crucial for differentiation. Also, ensure that the autologous serum used doesn't contain any factors that might cause cell detachment. Lastly, be gentle during media changes to avoid disturbing adherent cells, and ensure that the incubator's temperature, humidity, and CO2 levels are stable and optimal.

Your cells may also be exhausting media components. Try increasing the amount of medium to 1.5 ml/35mm well.

Hello Cemre,

Isolation of peripheral blood mononuclear cells Briefly, blood was diluted 1 : 1 with incomplete RPMI 1640 (iRPMI) containing 20 mm HEPES, 1 mm sodium pyruvate, 1 g/l sodium bicarbonate supplemented with 2 mm glutamine, 50 IU/ml penicillin and 50 µg/ml streptomycin . For density gradient separation of peripheral blood mononuclear cells (PBMC), 30 ml of diluted blood was layered onto 15 ml of Histopaque 1077 (Sigma-Aldrich) at room temperature. Following centrifugation at 400 g for 30 min PBMC were collected from the Histopaque interface, washed twice in 100 ml iRPMI and resuspended in 20 ml complete RPMI (cRPMI) (incomplete RPMI supplemented with 2% pooled heat inactivated human AB serum, SNBTS) for cell counting and culture. Cells were counted using a haemocytometer and white cell counting fluid (0·01% gentian violet and 3% acetic acid in water) and CD14+ cells enumerated by flow cytometry - donor dependant CD14+ range 5-12% .

Isolation of monocytes

Monocytes were isolated from PBMC by plastic adherence of 20–30 × 106 PBMC per ml in 5 ml cRPMI in 25 cm2 tissue culture flasks for 2 h in a humidified 37 °C incubator with 5% carbon dioxide (CO2). To remove non-adherent cells the overlying medium was aspirated and expelled over the adherent surface repeatedly and removed from the flask.

Adherent cells could be recovered by TripLE dissociation.

Blood needed to be less than 48 hours old for isolation of functional cells

Best wishes

Hi, did you ever figure out what the issue was? I am having issues with my differentiated macrophages adhering. I am extracting PBMCs by ficoll then doing negative selection of monocytes using a kit. I differentiate them for 5 days (using 20ng/mL of human recombinant M-CSF). I can see that they are differentiated (morphologically) but they are not adhering to the plate. I made sure I was using tissue culture treated plates and I even tried to add poly-d-lysine on the plates to help them adhere. Any suggestions?