Frequently Asked Questions

 

Technical Support:

Do I need to add l-glutamine to ESF 921 or ESF AF?

No, ESF 921 and ESF AF are complete media with no supplementation required for routine culturing.

Do I need to add antibiotics?

Expression Systems advises against the addition of antibiotics and/or fungicides as they may simply mask low level contaminations which can adversely affect growth and expression. There are no negative interactions between standard antibiotic supplements and ESF formulations.

How should I freeze my insect cells?

Insect cells can be frozen in ESF 921 or ESF AF with 10% DMSO. There is no need for the addition of fetal bovine serum. Please refer to the documents section for a detailed protocol on insect cell cryopreservation.

How many passages can I maintain my cells?

Expression Systems strongly supports a rigorous observation program of cell cultures rather than relying on a finite number of passages. Culture conditions such as temperature fluctuations, over-growing, under-seeding, oxygen deprivation and other environmental stresses play a far greater role than a count of passage times. Expression Systems considers a passage any time cells are diluted by the addition of media. A good rule of thumb is no more than 30 passages as this limits the number of environmental insults the culture has to withstand. Subtle morphological changes in the culture such as cell swelling, elongation or increased clumping are signals to initiate a new culture.

How do I use PBA?

Production Boost Additive should be added to the infected culture after infection has been established. For high MOI cultures, the PBA can be added 6-18 hours after infection. One strategy is to infect first thing in the morning and add the PBA at the end of the day. Alternatively the culture can be infected in the afternoon and PBA added the following morning. For low MOI cultures, the PBA should be added 24-36 hours post infection. PBA can interfere with virus uptake so it should not be added at the time of infection. PBA can be used between 1 and 10% of the culture volume, the exact percentage should be determined by titration. Please refer to the documents section for a detailed protocol on PBA.

How do I use Methionine-free media?

Methionine-free media is most commonly used for selenomethionine incorporation for X-ray crystallography. Methione is a non-essential amino acid for insect cells and therefore insect cell growth is not impacted by the absence of methionine. Please refer to the documents section for a detailed protocol for selenomethionine incorporation.

Is there EDTA/EGTA in ESF 921?

No, these chelating agents are not present in ESF 921 or ESF AF.

What is the NaCl content?

4.1 grams per liter.

Why do my cells look swollen after extended passage?

Chromosome instability in Sf9 cells leads to an accumulation of tetraploid cells. The increased genetic content leads to larger cells.Jarman-Smith,et al Biotechnol Prg 2002 May-Jun; 18(3):623-8. Doverskog et al (Biotechnol Prog. 2000 Sep-Oct;16(5):837-46.)have identified a cellular factor secreted by cells in lag phase that leads to this accumulation of tetraploid cells. Passaging of cells while still in log phase growth can lessen the effect Please see the documents section for the talk "Improving Reproducibility of Recombinant Protein Expression" for a demonstration of the effects of ploidy on protein expression. Click Here to view.

What kinds of cells are these?

Trichoplusia ni cells are derived from embryonic tissue of the cabbage looper. Spodoptera frugiperda cells are derived from ovarian tissue of fall armyworm. Sf9 cells are a subclone of Sf21 cells, and were selected for improved growth kinetics. S2 cells are derived from embryonic tissue of Drosophila melanogaster.