Search
Search
View additional product information for High Five™ Cells in Express Five™ Medium - FAQs (B85502)
29 product FAQs found
看起来这些细胞长得过满了。High Five细胞在有血清存在的条件下生长极为迅速。由于这一培养瓶中没有更多空间供细胞生长，因此请尽快分种这些细胞。我们建议您按照1:15的比例进行分种，并每天观察细胞生长情况。尽管可以加入20倍浓度的肝素，我们还是推荐您使用10 μg/mL这一工作浓度。尝试加入一些FBS（0.1%）10-20分钟，之后进行换液。
肝素可保存于室温。肝素通常以10 μg/mL的浓度进行使用，尽管超出20倍的浓度也是可行的。
我们同时推荐您以High Five和Sf9这两类细胞来小规模尝试蛋白转染生产实验。通常每毫升的Sf9细胞培养物所生成的蛋白产物稍多，因为它们可于3–4 x 10E6个细胞/mL的密度下完成转染。High Five在大于2 x 10E6个细胞/mL的密度下就已完全进入对数生长期，因此它们不宜在高于2 x 10E6个细胞/mL的密度下进行转染。
如果细胞密度过低，而且已经培养了4-5天，我们推荐您通过100 × g离心5分钟来富集细胞，并将其重悬于新鲜的培养基中。细胞在同一培养基体系中不应滞留超过4-5天，因为这一时间段内细胞会耗尽培养基中的养分，长时间暴露于较高温度下也会导致培养基自身发生降解。当培养时出现大量细胞碎片时，也可对细胞进行离心和富集。
昆虫细胞远比许多哺乳动物细胞系更为脆弱。这些细胞在过度生长和过度分离之后，会比哺乳动物细胞出现更多损伤。在悬浮培养时一定不要让细胞密度超过8 x 10E6细胞/mL或以低于0.5 x 10E6个细胞/mL的密度下生长。昆虫细胞需要略高于哺乳动物细胞的渗透压条件（340 μOsM）。昆虫细胞需要大量的O2，特别是在蛋白表达阶段。昆虫细胞培养基比哺乳动物培养基要酸得多（pH6.0-6.4）。昆虫细胞培养基是基于磷酸缓冲体系的。因此，无需使用CO2来维持pH值。
我们推荐采用Sf9或Sf21细胞进行重组病毒的转染，纯化和扩增操作。Sf9细胞的大小比较规则，易于操作，并能够生长为适于噬斑测定的优良单层细胞。Sf9与Sf21细胞也可用于重组蛋白的表达，不过使用High Five细胞系可能会获得更高的产量。
我们推荐使用High Five细胞系来表达分泌型重组蛋白。它们适于在无血清培养基中悬浮培养，可高水平表达的重组蛋白（Davis et al., 1992）
注意：一般来说，在优化蛋白表达情况之前，使用一种细胞系进行表达尝试比较方便。一旦您确定了您的重组蛋白已成功表达，即可尝试使用其他细胞系来优化表达水平。
不可，我们不推荐这样做。长期暴露于29 degrees C以上的温度会导致细胞死亡。这些细胞最好生长于27 degrees C或室温条件下。
不需要，昆虫细胞培养不需要CO2交换。
在转染和噬斑实验之前，细胞需要均匀地分布于组织培养板表面。这一操作能够确保：
-细胞不会分布不均匀而导致形成不对称的单层细胞。
-为转染操作提供最大的细胞表面积。
为了使细胞尽可能分散：
1.用手前后缓慢振荡培养瓶或平板，之后左右振荡。
2.重复以上操作四次，仔细查看以确保液体到达生长表面的所有区域。
3.请勿通过环状摇动来分散细胞，因为这将导致细胞聚集在平板边缘周围，而非均匀分布。
请参加下面关于昆虫细胞种类，培养基和相应的倍增时间（小时）的相关信息：
D.mel-2细胞；Schneider果蝇培养基+10% FBS(经热灭活)；18至24
High Five细胞；Express Five SFM培养基；约24
Sf9细胞；Sf-900 II SFM培养基；24至30
Sf9细胞；Sf-900 III SFM培养基；24至30
Sf21细胞；Sf-900 II SFM培养基；24至30
Sf21细胞；Sf-900 III SFM培养基；24至30
请参见下列关于细胞种类，培养基与细胞大小（微米）的相关信息：
D.mel-2；Schneider果蝇培养基+10% FBS(经热灭活)；10至12
High Five细胞；Express Five SFM；17.5至19.5
Sf9 细胞；Sf-900 II SFM；15至17.5
Sf9 细胞；Sf-900 III SFM；15至17.5
Sf21 细胞；Sf-900 II SFM；15至17.5
Sf21 细胞；Sf-900 III SFM；15至17.5
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.
Insect cells do not require CO2 or high humidity to grow, they can grow in a lab drawer at room temperature. We recommend this so people don't waste CO2 and other resources necessary for maintaining a tissue culture incubator. It should be noted, however, that the cells will grow in a humidified incubator.
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.
We recommend a density of 3.0x10E6 cells/vial for adherent cultures and 10-15x10E6 cells/vial for suspension cultures.
L-Glutamine is an essential nutrient in cell cultures for energy production as well as protein and nucleic acid synthesis. In general, insect cell media contain higher L-Glutamine levels than serum-free media for mammalian cells, primary cells, stem cells, etc. This is to support the fast metabolism of insect cells, especially when used for virus production and protein expression applications.
Note that removal of L-Glutamine from the media formulation aids in product shelf-life by preventing glutamine degradation during storage.
The biosafety level (BSL) for High Five Cells in Express Five Medium is typically biosafety level 2. Please note that this can vary from country to country.
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.
Heparin solution can be stored at room temperature. Heparin is usually added at 10 µg/mL although you can use it at 20 times higher concentration.
We recommend that both High Five cells and Sf9 cells be tried in small-scale infections for protein production. Sf9 cells often produce just as much protein per milliliter of culture because they will be infected at 3-4 x 10E6 cells/mL. High Five cells are definitely going into log phase at greater than 2 x 10E6 cells /mL; they should not be infected at a cell density higher than 2 x 10E6 cells/mL.
High Five cells are difficult to transfect and we typically get efficiencies of 5-13%. Other cell types are 40-50%. Also, High Five cells at confluence typically do not smoothly cover the whole surface of a flask. They still look a little patchy. They are confluent when they start to overlap and pile on each other.
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.
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.
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.
No, this is not recommended. Prolonged exposure to temperature higher than 29 degrees C will cause cell death. It is better to grow the cells at 27 degrees C or room temperature.
No, CO2 exchange is not required for insect cell culture.
Prior to performing transfections and plaque assays, cells need to be evenly distributed over the surface of a tissue culture plate. This ensures that:
- Cells do not distribute unevenly, leading to asymmetric monolayers.
- Maximum cell surface area is available for infection.
To disperse cells:
1. Rock the flask or plate slowly by hand forward and backward, then side-to-side.
2. Repeat this four times, watching carefully to ensure that the liquid reaches all areas of the growth surface.
3. Do not use a circular motion to disperse the cells, because this causes a concentration of cells around the edges of the plate rather than an even distribution.
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
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