Cell culture: considerations before you start

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Temperature	✓	36–37°C for mammalian cells.
	X	Temperature variability >0.5°C in the incubator.
CO2 level	✓	4–8% if the medium uses CO2-bicarbonate buﬀers to maintain optimal pH.
Health check	✓	Check cell culture on a regular basis – visually and under a brightﬁeld microscope.
	X	A sudden change in medium color (when pH indicators are used) and/or clarity or a foul smell may indicate infection.
	✓	A brightﬁeld microscope with 20–60x magniﬁcation will allow you to spot developing bacterial/fungal infections.
	X	In case of infection decontaminate and discard cells and clean the working area. The removal of infection with antimicrobial agents should be used as a last resort if no cell banks of the cell line exist.
Viability check	✓	Check cell culture on a regular basis – visually and under a brightﬁeld microscope.
	X	Sudden detachment/rounding up of adherent cells, excessive clumping of suspension cells, or the presence of cell debris is indicative of apoptosis.
Cell density	✓	Plate cells at 10–30% density during standard passaging. Plating cells too sparsely may impede their growth.
	✓	Cells should be passaged at 80–90% density, toward the end of the logarithmic growth phase.
	X	Do not allow your cells to overgrow – this will slow down their recovery after passaging.