• DNA conc for hTERT RPE-1 transfection: Typically 0.5–2 µg per well (6-well plate); optimize based on reagent and cell confluency.
• Opti-MEM: Preferred for transfection due to low serum & better uptake. Using 2% DMEM/F12 is not ideal but can work in a pinch; however, efficiency may drop.
• Cilia induction: Switching to 0% serum DMEM/F12 after 24 hrs is correct, but inefficient transfection or stress from suboptimal media could affect ciliogenesis.

try this

• Try reverse transfection or Lipofectamine 3000 if you haven’t already.
• Use serum starvation for 24–48 hrs post-transfection in complete DMEM/F12 without serum.
• Check cell density—RPE-1 needs ~70–80% confluency before starvation for good ciliogenesis.
• If available, get back to Opti-MEM or serum-free media during transfection for better results.

@Vikas manikrao shukre

We usually use lipo3000. and i transfect at almost 50% confluency. But I feel that the transfected cells are being lifted off.

Why cells are lifting off with Lipo3000

• Low confluency (50%) – try 70–80%.
• Too much reagent/DNA – reduce Lipo3000 and DNA amounts.
• Media stress – use Opti-MEM for transfection, then switch back to complete media after 4–6 hrs.
• Cell sensitivity – some lines detach easily.