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100% confluent monolayer (confluent monolayer) Monolayer has to be divided immediately. Too dense to be used for experiments. 90% confluent (sub-confluent) monolayer Ideal for protein expression studies. Too dense for transfections. Monolayer has to be divided the same day in case it is not used for experiments. To obtain sub-confluent monolayers seed about 2 ml of suspension culture with density 1x106 cells/ml into 8.8 cm2 Petri dish and leave the cells to settle for 45 min. Add more cells if monolayer appears too sparse. If the monolayer appears too dense, shake the dish to dislodge some cells and change the medium. It is difficult to seed desired monolayer densities from the first attempt as hemocytometer counts are approximate. 50% confluent (semi-confluent) monolayer Ideal for transfections. Can be used for generation of high titer P1 baculovirus stock. Too sparse for protein expression studies, but could become sub-confluent after overnight incubation at 28oC, and then used for protein expression studies. Note that some cells are flattened and spindle-shaped, which is a sign of healthy cell culture. To obtain semi-confluent monolayers seed about 2 ml of suspension culture with density 5x105 cells/ml into 8.8 cm2 Petri dish. Do not use less then 1.5 ml of cells to seed monolayers, as the monolayers will be very uneven, much more dense at the edges than in the middle. Add or remove cells as described above to achieve desirable density. 25% confluent monolayer Can be used for generation of high titer P1 baculovirus stock. 10% confluent monolayer Can grow to confluency in vent/close caps T-flasks, but not in Petri dishes or plates. Too sparse to be used for experiments. |