What is my cell type?

Endocytic capacity is the main axis. Transformed lines internalise large volumes of membrane per hour; primary lymphocytes in circulation have almost no basal endocytosis until activated. Reagent concentration that saturates a HeLa monolayer may barely engage a resting T cell.

Membrane composition differs too. Cholesterol-rich, ordered membranes resist fusion with lipoplexes, while highly fluid membranes fuse readily. This is why helper-lipid choice and even the culture's fatty-acid content can change results between labs using the same kit.

Immune sensing is the third axis and the one most often ignored. Primary cells express cytosolic DNA and RNA sensors; introducing plasmid DNA or unmodified mRNA can trigger type I interferon responses that kill the culture or silence the construct — a delivery failure that looks like biology.

Reagent vendors optimise on low-passage cells. Endocytic activity falls with passage and the membrane composition drifts, so a protocol that gives 90% efficiency at passage 5 can give 40% at passage 30. Define a working range — commonly 3–15 passages from thaw for HEK293 — and freeze fresh working stocks rather than passaging indefinitely.

If several lipid reagents fail in the same primary cell type, the problem is the delivery route, not the formulation. Move to nucleofection. If viability, not efficiency, is the limiting factor, move to a low-toxicity physical method such as magnetofection, or accept a lower payload dose in a gentler formulation.