flashBAC Manual - Oxford Expression Technologies

Table 1.Insect cell densities in suspension cultureCell line&MediumSf9 inserumfreemediumSf21 inserumcontainingmediumSeeding cell density(cells/ml -1 )0.3 - 0.5 x 10 6 6.0 x 10 6Maximum celldensity (cells/ml -1 )before passaging0.1- 0.2 x 10 6 2.0 - 2.5 x 10 610.2 Sub-culturing cells in suspension culturesUse aseptic technique throughout and work in a Laminar FlowCabinet or Class II Hood.Counting cells and determining cell viabilityBefore passaging cells, take a sample (about 1 ml into a 35 mmdish) to observe under the phase-contrast microscope usingx10 and x40 objectives. Healthy cells should look bright, roundand refractile. Many cells should also be in the process ofdividing into daughter cells.Using a second sample, count the cells using an electronic cellcounter or using a Neubauer counting chamber (Figure 5), asfollows. Using a Pasteur pipette or capillary tube, load asample into the counting chamber using capillary action only,i.e. avoid damaging the cells by forcing them through thepipette. Count all the cells within the 5 x 5 square grid on theOXFORD EXPRESSION TECHNOLOGIES 50