Reichert bright line hemacytometer

For anchorage dependent cellular growth, cells of different genotypes were seeded in 4 replicate wells at a density of 1×10⁴ cells per well in 6-well plates. At days 2,4 and 6 after seeding, cells were washed with 1x PBS, trypsinized, mixed with 0.4% Trypan blue solution (Sigma Aldrich) and then counted using the Reichert Bright-Line Hemacytometer (Hausser Scientific) by Trypan blue exclusion principle. For anchorage independent cellular growth, cells of different genotypes were grown in 4 replicate wells in two layers 6-well plates: The lower layer consisted of 0.6% (w/v) soft agar and the upper layer contained 1×10⁴ cells per well mixed in 0.3% (w/v) soft agar. Both soft agar layers consisted of 20% FBS. After 14 to 21 days, grown cellular colonies were scored.

Cells were seeded in triplicate in 12-well plates and then transfected the following day with siRNAs or overexpression vectors. One day following transfection, cells were incubated overnight in medium with or without 10% FBS and after 24, 48 and 72 hours after the medium change cells were washed twice with 1x PBS, trypsinized, mixed with 0.4% Trypan blue solution (sigma Aldrich) and then counted using the Reichert Bright-Line Hemacytometer (Hausser Scientific) by the Trypan blue exclusion principle.

Cell size was determined for eukaryotic species from micrographs of bright‐field images collected with a Zeiss AX10 microscope and recorded with an AxioCam ERc5s camera (Zeiss, Jena, Germany). A minimum of 50 micrographs per species were analyzed in ZEN software from Zeiss, and cell size determined from cross‐sectional area as the average cell diameter (ACD), calculated as for a circle. The scaling on the micrographs was calibrated using the lines on a Reichert Bright‐Line hemacytometer (Hausser Scientific, Horsham, PA, USA).