Manual / Product Insert

High Five Serum-Free Medium

Version: Version B

Manual / Product Insert

User Guide: Cellfectin II Reagent

Version: MAN0007821 Rev. 2.0

Citations & References

The type XIII collagen ectodomain is a 150-nm rod and capable of binding to fibronectin, nidogen-2, perlecan, and heparin.

  • Authors: Tu Hongmin; Sasaki Takako; Snellman Anne; Göhring Walter; Pirilä Paivi; Timpl Rupert; Pihlajaniemi Taina;
  • Journal: J Biol Chem (2002) 277:23092-23101
  • PubMed ID: 11956183

Citations & References

Type XIII collagen and some other transmembrane collagens contain two separate coiled-coil motifs, which may function as independent oligomerization domains.

  • Authors: Latvanlehto A, Snellman A, Tu H, Pihlajaniemi T,
  • Journal: J Biol Chem (2003) 278:37590-37599
  • PubMed ID: 12832406

Product Literature

HyCell CHO Medium: Targeted for high cell density and productivity across a broad variety of CHO clones

Product FAQ

Why is the glutamine requirement so high for Express Five® media?

Answer

The glutamine concentration is correct. Insect media have historically been 2 to 10 fold higher in glutamine concentration vs. classical mammalian media. Express Five SFM was optimized for High Five cells which have especially high requirements for glutamine and asparagine. The liquid basal media is actually 1.09x in concentration and was designed to be diluted to the correct 1x composition with 90 ml/L of the 200 ml L-glutamine. Because the L-glutamine concentration is so high, we left it out of the basal formulation to avoid problems associated with glutamine degradation and the resulting ammonia build-up which can be toxic.

Answer Id: E3050

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Product FAQ

My High Five cells are in log-phase growth in Grace’s Insect Medium plus serum. Why are the High Five cells forming large, free-floating clumps with very few cells attached?

Answer

The cells are most likely getting too confluent. High Five cells grow extremely fast in serum. Split the cells as soon as there is no more room on the flask for the cells. We suggest doing a 1:15 split and observing cell growth each day. Add heparin at 10 µg/mL, although it can be used at 20 times higher concentration. Try adding some FBS (0.1%) for about 10-20 minutes then change the media.

Answer Id: E11958

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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

Is there any cholesterol in your serum-free media for High-Five Cells?

Answer

There is no added cholesterol, however, there are yeastolates and hydrosylates which are cholesterol precursors. This is common for all insect serum-free media.

Answer Id: E11951

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Limited Use Label License

High Five™ Cells in Express Five™ Medium

Limited Use Label License
561
Catalog #

Product FAQ

How does one get High Five cells that have been adapted for suspension culture to adhere to flasks? This can be important when generating stable cell lines with High Five cells.

Answer

To get High Five cells that have been adapted for suspension culture to adhere to a flask for selection of stable clones, it is recommended to check for heparin in the media (heparin helps keep the cells in suspension) and add 0.1% FBS to the culture for 10-20 minutes, then change the media. Keep in mind that cells which have adapted to suspension culture often take time to revert to adherent culture. Maintain the cells in the same flask over a few passages under selection, each time passaging at 1:4 or 1:5, without spinning the cells down. This should select for the adherent cells.

Answer Id: E3308

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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

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

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

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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