Hi, I have experience with growing macrophages from buffy coats (1.4 M/mL PBMC, monocytes isolated by adhesion and washed) by differentiation in macrophage-SFM with GM-CSF. Now I need to establish dendritic cell cultures. I need quite many of them (counted in plates rather than milliliters). I heard it is not possible to generate good DCs from monocytes isolated only by adhesion, yet many protocols I observe online suggest exactly this - what is the best choice? Medium was RPMI, serum 10 %, antibiotics, glut, GM-CSF and IL4, changed every day, weekend double portion.

1) I now used CD14 magnetic beads (invitrogen) on buffy coat to isolate monocytes, but the yield was only about 1 %, way too few cells obtained. Cells were semi-adherent, I was recycling the non-adherent during media change.

2) Cells grew and slowly differentiate, many have irregular shapes, but rather roundish.

3) On day 6 I added LPS and TNF to induce maturation. Already after 4 h (and further after 24 h) majority of cells adhere and adopt stellate/elongated shapes, not fully confluent but density looks ok but there are some weird clumps of cells that look like sticking to something, perhaps some particulate matter. But I heard mature human DCs should not adhere but rather float.

I would appreciate clarifications from more experienced users. I need simplest protocol possible. Also the need for magnetic isolation with CD14 seems to be an issue - some people say it is critical for purity of cells, some protocols rely only on adhesion. Can someone comment or even send a protocol?