Product FAQ

I performed an LR reaction, followed by transfection into Sf9/Sf21 insect cells, but do not see any signs of infection even though it’s been 72 hours. What should i do?

Answer

Please see our recommendations below:

- Check the LR reaction by PCR analysis prior to transfection into insect cells.
- We recommend using Grace’s Insect Cell Culture Medium, Unsupplemented during the transfection experiment instead of serum-free medium, as components in serum-free medium may interfere with transfection.
- Ensure that FBS, supplements, or antibiotics are not included during transfection, as the proteins in these materials can interfere with the Cellfectin II Reagent.
- Use the LR recombination reaction using the pENTR/CAT plasmid as a positive control and Cellfectin II Reagent only (mock transfection) as a negative control.
- Ensure that cells are in the log phase of growth with >95% viability, and the amount of cells are in accordance with the suggestions in the manual.
- Cells may not show signs of viral infection for up to a week depending on transfection efficiency; continue culturing and monitor cells daily for signs of infection.

Answer Id: E9459

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Product FAQ

What methods can be used to detach tightly adherent Sf9 and Sf21 cells cultured in Grace’s media supplemented with 10% heat-inactivated FBS, Sf-900 II SFM, or Sf-900 III SFM?

Answer

Sf9 and Sf21 cells should be lightly adherent cells. However, there are some Sf9 and Sf21 cells that attach to culture vessels very tightly. The use of enzymes such as trypsin, collagenase, hyaluronidase, TrypLE Express, and TrypLE Select have been tried without success for passaging cells. The main problem is that the cells do not attach well after having dissociated with the enzymes.

The best method to use is to culture cells in a T-flask. Close cap tightly and hold flask with cap pointing towards the ceiling. Hit the bottom of the flask over a counter 2-3 times with medium force. Cell detachment may be 60-80% and not 100%. This will allow for detachment of enough cells for passaging. If tapping the flask over the counter is performed with too harsh of a force or too many times, cell viability will be greatly affected.

If possible, we recommend that you culture cells in suspension conditions. Cells in suspension cultures can be passaged directly into adherent conditions when needed. The culture of cells in suspension conditions will allow for higher cell densities as cell growth is not limited to the surface area.

Answer Id: E11939

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Product FAQ

What medium do you recommend to grow Sf9 or Sf21 cells for performing plaque assays?

Answer

We would recommend using our Grace’s Supplemented Insect Medium (Cat. No. 11667037) or SF-900 Medium (1.3X) (Cat. No. 10967032). Grace’s Supplmented Insect Media is a 2X formulation intended for use in the agarose overlay when performing the plaque assay. The media has been modified with:
- Lactalbumin hydrolysate
- Yeastolate
- L-glutamine
- Sodium bicarbonate

Please go here for the complete formulation (http://www.thermofisher.com/us/en/home/technical-resources/media-formulation.72.html). SF-900 Medium (1.3X) is a complete serum-free low protein insect cell culture medium for Sf9 and Sf21 cells, which can be used for 1% agarose overlays used for plaque assays.

Answer Id: E11947

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Product FAQ

What are the sizes of Sf9, Sf21, High Five cells, and D.mel-2 cells?

Answer

Please see below for information related to type of cells, media, and cell size in microns:

D.mel-2; Schneider’s Drosophila + 10% FBS heat-inactivated; 10 to 12
High Five cells; Express Five SFM; 17.5 to 19.5
Sf9 cell; Sf-900 II SFM; 15 to 17.5
Sf9 cells; Sf-900 III SFM; 15 to 17.5
Sf21 cell;s Sf-900 II SFM; 15 to 17.5
Sf21 cells; Sf-900 III SFM; 15 to 17.5

Answer Id: E11931

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Product FAQ

What is the procedure to thaw frozen insect cells?

Answer

The following protocol describes a general procedure for thawing cryopreserved cells. For detailed protocols, always refer to the cell-specific product insert.

1. Remove the cryovial containing the frozen cells from liquid nitrogen storage and immediately place it into a 37°C water bath.
2. Quickly thaw the cells (< 1 minute) by gently swirling the vial in the 37°C water bath until there is just a small bit of ice left in the vial.
3. Transfer the vial into a laminar flow hood. Before opening, wipe the outside of the vial with 70% ethanol.
4. Transfer the desired amount of pre-warmed complete growth medium appropriate for your cell line dropwise into the centrifuge tube containing the thawed cells.
5. Centrifuge the cell suspension at approximately 200 x g for 5-10 minutes. The actual centrifugation speed and duration varies depending on the cell type.
6. After the centrifugation, check the clarity of supernatant and visibility of a complete pellet. Aseptically decant the supernatant without disturbing the cell pellet.
7. Gently resuspend the cells in complete growth medium, and transfer them into the appropriate culture vessel and into the recommended culture environment.

Note: The appropriate flask size depends on the number of cells frozen in the cryovial, and the culture environment varies based on the cell and media type.

Answer Id: E5549

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Product FAQ

How can I concentrate my insect cells to increase the cell density?

Answer

If the cell density is too low and the cells have been in culture for 4-5 days, we recommend concentrating the cells by centrifuging them at 100 X g for 5 minutes and resuspending them in fresh medium. Cells should not be left in the same medium for more than 4-5 days as nutrients in the medium will have been used up by the cells in that period, and the medium itself degraded due to prolonged exposure to warm temperatures. Cells should also be centrifuged and concentrated if a lot of cell debris is observed in culture.

Answer Id: E11938

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Product FAQ

Which insect cell line is best to use?

Answer

We recommend Sf9 or Sf21 cells for transfection, purification, and amplification of recombinant virus. Sf9 cells are regular in size, easy to manipulate, and form good monolayers for plaque assays. Sf9 and Sf21 cells can also be used for expression of recombinant proteins, but the High Five cell line may produce higher levels. We recommend the High Five cell line for expression of secreted recombinant proteins. They are grown in serum-free medium, adaptable to suspension culture, and produce high levels of recombinant protein (Davis et al., 1992; see http://www.ncbi.nlm.nih.gov/pubmed/1368794).

Note: Generally it is easier to use one cell line for procedures up to optimization of protein expression. Once you have confirmed expression of your recombinant protein, other cell lines can be tried for optimization of expression levels.

Answer Id: E11936

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Product FAQ

What are the main differences between insect cell culture and mammalian cell culture?

Answer

Insect cells are much more fragile than a lot of mammalian cell lines. They suffer much more damage than mammalian cells from overgrowth and over-splitting. Never let cells go above 8 x 10E6 cells/mL or grow at densities less than 0.5 x 10E6 cells/mL in suspension. Insect cells require a little more osmotic pressure than mammalian cells (340 µOsM). Insect cells use a lot of O2, especially during protein expression. Insect cell culture media is more acidic than mammalian media (pH 6.0-6.4). The insect cell culture media is phosphate buffer based. Therefore, no CO2 is needed to maintain the pH.

Answer Id: E11937

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Product FAQ

Is it necessary to heat-inactivate my serum before adding it to my medium?

Answer

Heat inactivation is not necessary. Our team has routinely used serum that has not been heat-inactivated, and we have not observed any effect on cell growth or morphology.

Many cells do not require heat-inactivated FBS. Some cells prefer heat-inactivated FBS. For instance, we use heat-inactivated FBS for our insect cell lines, i.e., Sf9 and Sf21 cells.

Answer Id: E11945

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Product FAQ

What are the doubling times for robust growth and maintenance of different insect cell lines?

Answer

Please see below for information related to the type of cells, media, and doubling time in hours:

D.mel-2; Schneider’s Drosophila + 10% FBS heat-inactivated; 18 to 24
High Five cells; Express Five SFM; approximately 24
Sf9 cells; Sf-900 II SFM; 24 to 30
Sf9 cells; Sf-900 III SFM; 24 to 30
Sf21 cells; Sf-900 II SFM; 24 to 30
Sf21 cells Sf-900 III SFM; 24 to 30

Answer Id: E11932

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Product FAQ

How is ExpiSf CD Medium different from Sf-900 II SFM or Sf-900 III SFM?

Answer

All three media formulations are protein-free and serum-free and optimized for Sf9 and Sf21 cell culture and protein expression. However, ExpiSf CD Medium is a chemically defined, yeastolate-free formulation whereas both Sf-900 II SFM and Sf-900 III SFM media contain hydrolysates, therefore making them undefined formulations. The absence of hydrolysates or any other undefined component makes ExpiSf CD Medium a superior formulation with greater lot-to-lot cell growth and protein expression consistency run after run. In terms of maximum cell densities, ExpiSf CD Medium can support the highest maximum Sf9 cell density of approximately 20 x 10E6 cells/mL whereas Sf-900 II SFM and Sf-900 III SFM support maximum Sf9 cell densities of 10 x 10E6 cells/mL and 14 x 10E6 cells/mL, respectively.

Answer Id: E16212

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Product FAQ

Can I adapt my own insect cell line for growth in ExpiSf CD Medium?

Answer

Yes, you may be able to adapt your Sf9 cells for growth in ExpiSf CD Medium. We recommend following a serial adaptation protocol to slowly acclimate your cells for growth in ExpiSf CD Medium. A detailed cell adaptation protocol is provided in the ExpiSf CD Medium User Guide (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017561_ExpiSfExpressSystem_SmartStart_UG.pdf). Long-term adaptation in ExpiSf CD Medium may increase productivity of your Sf9 cells, and should support high-density growth. However, there is no guarantee that other insect cell lines will achieve the same levels of baculovirus and protein expression as the ExpiSf9 Cells. In limited testing, we have found other Sf9 and Sf21 cell lines to achieve lower maximum cell densities when fully adapted to ExpiSf CD Medium with lower protein yields. The performance of your own insect cell line in ExpiSf CD Medium will need to be empirically determined.

Answer Id: E16211

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Product Literature

Bac-to-Bac

Product Literature

Brochure: Gibco basal medium chart (Japanese)