HEKPlus Protein Expression System (ATCC® ACS-4800-K)

Subcultivation 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.
Application Protein Expression
Biosafety Level 2
[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.]
Comments 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.
Product Format 1 Kit
Basic Documentation
  1. HEKPlus Protein Expression System Parameters
    Cell density at transfection, ratio of DNA to GeneX
    Date Updated: 3/27/2014
  2. GeneXPlus transfection reagent
    Date Updated: 3/27/2014
  3. HEKPlus Protein Expression System vectors
    Expression vectors containing the SV40 origin of replication should be used. These vectors permit more plasmid copies to persist in the transiently transfected cells.
    Date Updated: 3/27/2014
  4. HEKPlus SFM supplements
    Complete culture medium is prepared by adding L-glutamine to the HEK
    Date Updated: 3/27/2014
  5. HEKPlus Protein Expression System time for optimal protein expression
    It usually takes 2 to 3 days to see optimal expression of intracellular proteins and 6 days for optimal expression of secreted proteins. It is recommended that a time course be performed for...
    Date Updated: 3/27/2014
  6. Advantage of using HEKPlus Protein Expression System
    The HEK
    Date Updated: 3/27/2014
  7. HEKPlus Protein Expression System advantages
    The HEK
    Date Updated: 3/27/2014
  8. HEKPlus Protein Expression System to express hard-to-express proteins
    Yes. The HEK
    Date Updated: 3/27/2014
  9. HEKPlus Protein Expression System Scale
    The HEK
    Date Updated: 3/27/2014
  10. HEKPlus Protein Expression System secreted proteins
    It is recommended to collect cell supernatants at day 6 post-transfection. However, one may want to perform a time course study to determine optimal time for collection post-transfection for each...
    Date Updated: 3/27/2014
  11. HEK293T/17 SF cells are clumping
    No. Small aggregates are easily dispersed with minimal pipetting and do not affect the performance of the cell line.
    Date Updated: 3/27/2014
  12. Replenishing medium for HEK293T/17 SF cells
    Yes. Incomplete media changes affect the cell viability and recovery. Spent medium should be completely removed and replaced with fresh medium every 2 to 3 days.
    Date Updated: 3/27/2014
  13. Media volume for HEK293T/17 SF cells
    For protein expression, it is important to have adequate shaking in the culture vessel. We usually add culture medium to 20% volume of a shaker flask.
    Date Updated: 3/27/2014
  14. HEKPlus Protein Expression System Production
    Yes. Our validation data indicates that the ATCC® ACS-4800-K ™ HEK
    Date Updated: 7/24/2014