Subculture cells at log phase (when cells are ready for passaging, i.e., every 2-3 days, and are approximately 2 x106 cells/mL). Pre-warm fresh growth medium prior to use.
Note: Slight aggregates may be observed, but they are easily dispersed with minimal pipetting and do not impact the performance of the cell line.
1. Swirl the flask gently to evenly distribute cells in medium. Remove a small volume of cells from the flask and perform cell count.
2. Determine the total number of cells, viable number of cells, and percent viability using a hemacytometer or cell counter (e.g., Vi-cell Beckman Coulter Counter).
3. Determine the volume of growth medium needed to subculture. Cells should be seeded at a density of 5 x 105 cells/mL (for maintenance) or 8 x 105 cells/mL (for next-day transfection).
4. Centrifuge the cell suspension at 170 × g for 5 minutes. Carefully aspirate the supernatant and discard.
5. Resuspend the cell pellet in fresh growth medium at a density of 5 x 105 cells/mL (for maintenance) or 8 x 105 cells/mL (for next-day transfection). Dislodge cell pellet by gently pipetting cells in the medium.
6. Return flasks to the shaking incubator. Cells must be passaged at least twice prior to transfection to ensure optimal viability.
Replace medium every 2 to 3 days.
[It is the responsibility of the investigator to determine appropriate safety procedures for use with this material. As a reference, laboratory safety is discussed in the publication Biosafety in Microbiological and Biomedical Laboratories and can be accessed by searching "BMBL" at www.cdc.gov.]
HEK293T/17 SF suspension cells, ATCC ACS-4500 are pre-adapted to HEKPlus SFM, ATCC-ACS-4002 and can be cultured in suspension and directly transfected in HEKPlus SFM using GeneXPlus transfection reagent, ATCC ACS-4004. Transfection complexes can be prepared in the HEKPlus/ medium as well. Boost reagent has been shown to significantly enhance extracellular protein expression levels in cultures at volumes greater than 5 mL.
The HEKPlus Protein Expression System supplies sufficient reagents for the transfection of the equivalent of 500 mL of culture or 1 x 109 total cells (e.g. 16 x 30-mL reactions, 50 x 10-mL reactions) at an optimized transfection reagent ratio of 1:2 for DNA (µg) to transfection reagent (µL). The reagent volumes can be easily scaled up, if large-scale protein production is desired.