How to do mammalian cell culture? Behzad Khoshnood, 2015

General information Types of cell lines Cells cultivated directly after being isolated from organism: primary culture (limited lifespan) Cell lines have unlimited lifespan: immortalized To become a cell line, mutations have to occur; E.g : in the telomerase, anti-apoptotic genes, …  Type of mutation must be kept in mind True for all cell lines: DON’T VORTEX mammalian cells!

Prepare yourself Put on a clean lab coat and fresh gloves!

Prepare the hood Open the hood, start ventilation and allow air flow to build up! Desinfect the entire interior of the hood with 70 % EtOH!

Bring all items you will need in the hood Spray every single item (incl. Pipettes, media flasks,...) before bringing it in the hood with 70 % EtOH! Try to avoid going in and out of the hood by planning your consumption of media and plastic wear!

Don’t mess with the air flow Don’t block the airflow by putting items on the grid (vertical flow)! Always bring as few items in the hood as possible (both)!

Bring your cells and pre-warmed medium Carry your cells very carefully to the hood! Avoid medium getting in contact with lid (cell culture flasks and well plates)! Keep orientation of flasks (bottle neck should point up)!

Keeping items sterile in the hood Avoid foam and bubbles of any kind! Be honest with yourself and don’t use doubtful items! Keep bottles and flasks covered or closed! Open long pipettes at the correct end (filter), attach to pipette aid, then remove cover!

Handle your cells carefully Don’t scratch into cell layers! Don’t add fridge-cold medium! Don’t vortex your cells! Don’t let them sit in the hood longer than necessary! Always cover your cells, if possible, even in the hood! Monitor your cells on a regular base (also color of medium)!

Once you are done... Bring your cells carefully to the incubator! Bring all your media back in the fridge! Remove waste and items that don’t stay in the hood! Clean with 70 % EtOH!

GOOD LUCK!