<p>Infection of Sf21 cells for protein production 1. Plate the cells from a large flask (T175) that’s confluent (about 100 %), but not yet over-confluent (contains about 38 ml medium, cell density usually about 1x106/ml, if in doubt count with hemacytometer). As a rule of thumb the cells should be appr. 60-80 % (ideally 70 %) when infected. The cells will adhere about 15-20 min after you plate them (check under microscope to be sure). In general, plate about 2.5x106 on a p60 (3 ml) and 9x106 (9 ml) on a p100. 2. During infection it’s important that the volume is minimal for this to be efficient. When cells are ready to infect, aspirate the medium and add the following: 200 – 500 µl virus stock + medium up to 2 ml final volume (p60) or 1 ml virus stock + 3 ml medium (p100). Infect for one hour at room temperature on a rocking table (not going too fast so the cells will dislodge). 3. Aspirate infection medium and replace with fresh medium (5 ml on p60, 10 ml on p100) 4. Harvest cells after appr. 48 hrs and analyze for protein expression.</p><p>Sf21 cells are grown at 27 ˚ C in Grace’s supplemented medium further supplemented with 50 µg/ml gentamicin and 10 % FCS.</p>