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View additional product information for Sf9 cells in Sf-900™ II SFM - FAQs (11496015)
40 product FAQs found
这种形态的细胞一般在细胞生长数周后才会出现。对于高密度的冻存细胞或表达蛋白而言，它们并不见得有什么害处。不过，如果这些细胞的比例很高，就可能提示细胞正在发生老化，应以一管新的冻存细胞重新开始培养。
黄色颗粒可能是细胞器，聚集物或细胞碎片。我们第一次融解冻存细胞时也看到过这些粒子。为了避免出现此种现象，您可将振荡培养物静置5分钟，之后将上1/3的培养物转移至一个新的培养瓶中，移种之前要对细胞进行计数。您也可使用最高 200U/mL的肝素来减少聚集。终浓度为0.2%的Pluronic溶液也可用于减少剪切力的作用，振荡速度也可增至100–120rpm。刚刚解冻的细胞也可能发生破裂并释放小型囊泡，正如显微镜在高倍放大后观察到的那样。为了减少这些小颗粒的数量，应对细胞进行快速而彻底的解冻操作，这对于成功融化冻存细胞十分重要。此外：
1.将冻存管从水浴中转移至超净台时，请将冻存管一直置于冰上。
2.尽可能温和地进行加样操作，因为较大表面积使细胞很容易受到剪切力的损伤。
3.从解冻后的冻存管向培养瓶中转移细胞时，不宜使用冷培养基。
4. 30-45分钟后大部分细胞一经贴壁，就应更换培养基。应使用预温的培养基（27 degrees C）进行更换。冻存培养基中所含的10% DMSO在长时间接触后（最高1小时）会杀死细胞。
5.最后，需要检查细胞是否被污染。将一小部分培养物接种于T-25培养瓶中培育三天，之后检查是否浑浊。
这些细胞稀释过度了。一般情况下，我们推荐Sf9细胞的最低培养密度为50万个细胞/mL。
这对于Sf9细胞来说很正常。通常情况下，我们推荐让细胞恢复一周时间，之后再检查细胞活力。这时细胞应接近1-2百万个细胞/mL的密度，可以分至更低浓度进行培养。
我们一般推荐您通过机械法，而非酶促消化法（即胰酶/EDTA）解离Sf9细胞。您只需用纸巾包裹培养瓶，再将其较窄的一端在台面敲击，即可达到这一目的。这一操作会使昆虫细胞从培养瓶壁脱离下来。
许多抗生素都适用于昆虫细胞。下列为常用抗生素：
青霉素/链霉素：50–100 U/mL；50–100 μg/mL
两性霉素B(Fungizone 抗真菌剂）：0.25 μg/mL
庆大霉素：500 mL培养基中加入0.5 mL 10 mg/mL溶液（最终浓度10 μg/mL）
热灭活操作不是必需的。我们的团队直接使用未经热灭活的血清，并未发现其对细胞生长或细胞形态有任何显著影响。
是的，我们提供各类无血清的昆虫培养基，请点击此处(https://www.thermofisher.com/us/en/home/life-science/cell-culture/insect-cell-culture/insect-cell-culture-misc/serum-free-media.html?icid=cvc-insect-media-c2t2)浏览我们所提供的培养基以及它们之间的区别。
无论何时对细胞进行计数操作，您都可通过台盼蓝分析来检查细胞活力。如果细胞的活力在约95%以上，并且每25小时左右完成一次倍增，就可以继续使用。如果活力下降而倍增时间延长，可转染能力就会受到影响。传代超过30次的细胞可能会出现老化迹象，蛋白合成随之延迟。
Sf9与Sf21细胞应是贴壁较弱的细胞。不过，某些Sf9和Sf21细胞能够非常紧密地贴附于培养容器。我们尝试使用过胰酶，胶原酶，透明质酸酶，TrypLE Express和的TrypLE Select，均未能成功传代细胞。几次传代之内细胞还可能会解离，但之后就无法有效解离了。
最佳的方法是在T-培养瓶中培养细胞。盖紧瓶盖，并以瓶盖朝上的方向抓住培养瓶，在台面上以中等力量敲击瓶底2-3次，将会有60%-80%的细胞解离下来，但不会达到100%，这样就有足够的细胞解离下来用于传代。如果在台面敲击培养瓶过于猛烈或次数太多，细胞活力将受到很大影响。
在可能条件下，我们推荐您以悬浮条件培养您的细胞。如果需要的话悬浮培养的细胞可随时直接传代至贴壁培养的条件。悬浮培养的细胞也能够达到更高的细胞密度，因为细胞生长不受限于表面积。
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.
In the U.S. we sell Sf9 cells which are adapted to SFM. The catalog number is 11496-015.
Yes, you can grow Sf9 cells in glass vessels. The only concern would be if your glass vessels are not clean enough and there may be residual detergent left which will hurt your cells.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
These cells appear after cultures have been grown for several weeks. These do not seem to be detrimental to plating of high titer stocks or expression. However, if they are in high numbers, it may indicate that the cells are becoming old and that the culture should be re-started with a new stock of frozen cells.
Yellow particles could be cell organelles, aggregates, or debris. We see this when we first thaw frozen cells. To avoid this, you can let the shaker culture sit for 5 minutes, then transfer top 1/3 to a new flask, making sure to count cells first. You can also use heparin at up to 200 U/mL to decrease aggregation. Pluronic solution at a final concentration of 0.2% can also be used to decrease shearing, and increase shake speed to 100-120 rpm. Recently thawed cells seem to be breaking up and releasing small vesicles, as observed under high magnification. To reduce the amount of those small particles, cells need to be rapidly but completely defrosted for successful thawing to take place. Also:
1. Place vial on ice during transfer from water bath to sterile hood.
2. Pipette as gently as possible because cells shear easily due to larger surface area.
3. Cells may not have been placed in cold media after removal from defrosted vial into flask.
4. Media may not have been changed after 30-45 minutes once a majority of cells had attached. Media change should be with pre-warmed media (27degrees C). 10% DMSO in freezing medium will kill the cells if left on them for long periods of time (1 hour seems to be a maximum).
5. Lastly, cells should be checked for contamination. To do so, plate a small portion of culture in a T-25 flask and incubate for 3 days, checking for cloudiness.
These cells were oversplit. Typically, we recommend the lowest density for the Sf9 cells to be 0.5 million cells/mL.
No, but the cells are still stressed. Determine the cell density and spin down the cells at 130 x g for 3 minutes and resuspend the cell pellet with fresh medium. If the cell density is at least 2 x 10E6 cells/mL, proceed by seeding cells at 0.4 x 10E6 cells/mL and transfer into a new flask (125 mL shake flask; cell culture volume: 30-50 mL). Let the cells grow for 2 days. If the cell viability is not at least 80% and the cell density is not at least 2 x 10E6 cells/mL, discard the culture and start thawing a new vial.
We typically recommend dislodging Sf9 cells mechanically rather than enzymatically (i.e., with trypsin/EDTA). To do so, wrap your flask with a towel, and bang the narrow end along the hood. This should cause the cells to dislodge from the flask.
Many antibiotics are suitable for use with insect cells. The following antibiotics are commonly used:
- Penicillin/Streptomycine: 50-100 U/mL; 50-100 µg/mL
- Amphotericin B (Fungizone antimycotic): 0.25 µg/mL
- Gentamicin: 0.5 mL of 10 mg/mL solution in 500 mL media (final concentration: 10 µg/mL)
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
Yes, we offer a variety of serum-free insect media. Please go here (http://www.thermofisher.com/us/en/home/life-science/cell-culture/insect-cell-culture/insect-cell-culture-misc/serum-free-media.html?icid=cvc-insect-media-c2t2) to view the media we offer and the differences between them.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
Whenever the cells are counted, a trypan blue assay should be performed to check for cell viability. If the cells are maintaining viability above approximately 95% and are doubling every 25 hours or so, then they are still okay to use. If the viability drops and the doubling time increases, the infectability will be affected. Cells older than 30 passages may start to show signs of aging with delayed protein production.
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.
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