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View additional product information for ExpiSf™ CD Medium - FAQs (A3767805, A3767804, A3767803, A3767802, A3767801)
36 product FAQs found
Protein yield can vary greatly from protein to protein. We strongly recommend using the pFastBac 1 - Gus positive control vector (included in the ExpiSf Expression System Starter Kit as well as sold separately - Cat. No. 10360014) to generate Gus-expressing baculovirus and express Gus recombinant protein. The typical yield of Gus protein using the ExpiSf Expression System is approximately 250,000 activity units/mL following the recommended quantification protocol described in the ExpiSf Expression System User Guide (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017532_ExpiSfExpressionSystem_UG.pdf). Quantification of the Gus positive control protein will help determine if the low protein yield is due to a low expressing protein, low baculovirus titer/MOI used, poor baculovirus stock quality, a problem with the system components, or transfection and cell culturing conditions. If the expected protein yield is not being achieved with the Gus positive control, we recommend checking the following:
1. Ensure that you have a healthy ExpiSf9 cell culture:
- Cells are >90% viable during normal passaging and at the time of transfection and infection.
- Cells exhibit a doubling time of approximately 24 hrs.
- Cells recover rapidly post-thaw (i.e., reach a cell density of 5 x 10E6 cells/mL with ≥80% viability within 4-5 days post thaw); if not, verify that the culturing guidelines provided in the product manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017532_ExpiSfExpressionSystem_UG.pdf) are being followed, and thaw a new vial of cells if necessary.
- Verify that the incubator temperature is set at 27 degrees C for your cultures as temperatures higher than 28 degrees C may cause cells to overgrow and display reduced yield capacity overtime. Verify that the shake speed is 125±5 rpm for shakers with a 19 mm or 25 mm orbital diameter and 95±5 rpm for shakers with a 50 mm orbital diameter. All of our shake speed recommendations are provided for Nalgene PETG non-baffled Erlenmeyer flasks; if a different culture vessel is being used, additional shake speed and/or other culture parameter(s) optimization may be required.
2. Ensure that you have a high-quality baculovirus stock:
a. Ensure that pure and high-quality bacmid DNA is used:
- Check the quality of the recombinant bacmid by agarose gel electrophoresis to confirm DNA integrity.
- Ensure that a single white colony is picked during bacmid preparation to avoid a mixture of recombinant and non-recombinant baculovirus.
- Confirm that pure bacmid DNA is used (i.e., contains only recombinant bacmid and no empty bacmid), consider screening other DH10Bac transformants (i.e., white colonies).
- Look for the typical signs of a good transfection reaction: When ExpiSf9 Cells are efficiently transfected and baculovirus is being produced, viable cell density is between 80-90% at Day 3 post-transfection and will decrease to <80% at Day 4 post-transfection.
b. Ensure that the recombinant baculovirus contains the gene of interest:
- Verify transposition of bacmid DNA by PCR analysis using the pUC/M13 Forward and Reverse primers.
- Re-transfect ExpiSf9 Cells with new recombinant bacmid DNA, if necessary.
c. We recommend using the suspension-based transfection protocol for generation of high-quality P0 virus, as multiple rounds of viral amplification following the classical adherent format can result in spontaneous excision of the gene of interest as well as the formation of defective viral particles.
d. Ensure that the baculovirus stock is stored properly: Baculovirus stock should be stored at 4 degrees C, protected from light for up to 12 weeks. Alternatively, baculovirus stocks can be frozen and stored at -80 degrees C or in liquid nitrogen (no DMSO or cryopreservative) for longer periods. We do not recommend repeated freeze/thaw cycles of your virus stock. Frozen virus should be stored in small aliquots and not re-frozen once thawed.
3. Ensure that the cells are seeded at 5 x 10E6 viable cells/mL at the time of ExpiSf Enhancer addition.
4. Ensure that the correct amount of ExpiSf Enhancer is added 18-24 hrs prior to infection. Incubating enhancer-treated cells for longer than 24 hrs may result in decreased infection efficiency and low protein yields.
5. Ensure that the cell density at the time of virus infection is at 5-7 x 10E6 viable cells/mL. Infecting cells at higher densities will lead to sub-optimal infection conditions and poor protein yields.
6. Ensure that the optimal volume of baculovirus stock is used to infect cells. We recommend starting with an MOI of 5 and further optimizing the MOI (from 3-10), if necessary.
7. If baculovirus titer wasn't determined, we recommend testing a series of virus stock dilutions to determine the optimal volume to use for virus infections. A starting dilution range of 1:50 - 1:100 (virus stock : culture volume) is a good starting point.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
We offer LabCoat Live: SmartStart Training for the ExpiSf Expression System (/content/lifetech/global/en/home/global/forms/labcoat-live-smartstart-training-expisf.html). LabCoat Live is a unique learning environment that allows you to gain hands-on experience in your lab by providing the necessary reagents and protocols, and offering lectures through a flexible and affordable online environment with a self-paced lab experiment.
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Yes. There is a system page at www.thermofisher.com/expisf with comprehensive product information. We also offer our ExpiSf LabCoat Live virtual training course that provides interactive online lectures. For a thorough understanding of the system, experiment setup, and data analysis, sign up at www.thermofisher.com/labcoatlive (check for availability in your region). These are excellent resources for getting started or for troubleshooting.
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The easiest way to get started is by purchasing the ExpiSf Expression System Starter Kit (Cat. No. A38841). This is an all-in-one kit that contains all of the reagents necessary to generate recombinant baculovirus and express your proteins. Reagents for cloning your gene of interest into the pFastBac Expression Vector are sold separately and can easily be found at www.thermofisher.com/expisf .
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ExpiFectamine Sf Transfection Reagent typically exhibits lower toxicity and a faster complexation time and has a simpler protocol compared to other DNA transfection reagents. Lower toxicity means that no media change is required post-transfection. Shorter complexation time and simpler protocol means that DNA:lipid complexes are formed in only 5 mins and undiluted DNA is added directly to diluted ExpiFectamine Sf solution. This translates to time savings and the streamlining of your transfection reactions. This can be particularly advantageous where a higher throughput is required.
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Yes. The ExpiFectamine Sf Transfection Reagent can be used to efficiently transfect Sf9 and Sf21 cells. The reagent can be used to transfect these cells in both adherent and suspension culture formats.
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The optimal complexation time is 5 mins. We have observed a gradual drop in protein yield if complexes sit for longer than this recommended time; if complexes are left to sit for 20 mins or longer, transfection efficiency will be drastically reduced (>50% reduction in baculovirus titer produced).
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No. The ExpiFectamine Sf Transfection Reagent is a low-toxicity reagent and is designed to be used without media exchanges. There is no need to remove transfection complexes or to change growth medium following transfection.
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Both adherent- and suspension-based transfection protocols will allow you to generate high-titer baculovirus stocks using the ExpiSf Expression System. You may use either protocol interchangeably depending on your lab capabilities and preference. The use of suspension culture versus adherent culture can increase the cell density per mL of culture, and therefore will allow you to generate a larger amount and volume of P1 virus stock in shorter period of time.
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ExpiSf CD Medium is a core component of the ExpiSf Expression System and has been designed to support high-density growth of suspension ExpiSf9 cells. The medium supports superior baculovirus generation and protein expression as part of the ExpiSf System. You may adapt your own Sf9 cell line for growth in ExpiSf CD Medium using a sequential adaptation protocol described in the ExpiSf CD Medium User Guide (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017561_ExpiSfExpressSystem_SmartStart_UG.pdf). However, there is no guarantee that other insect cell lines will achieve the same levels of baculovirus and protein expression as the ExpiSf9 Cells.
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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.
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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.
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The time of harvest is highly dependent upon the nature of the protein. Because baculovirus is a lytic virus, infected cells will eventually lyse. It is important to determine the expression kinetics for each protein, as many proteins (secreted or non-secreted) may be degraded by cellular proteases released in cell culture. Maximum expression is usually observed between 30 and 72 hrs post-infection for secreted proteins and between 48 and 96 hrs post-infection for non-secreted proteins. For some proteins, you may need to protect the recombinant protein from proteolysis by supplementing cultures post-infection and/or at the time of harvest with protease inhibitors. We recommend using Halt Protease Inhibitor Cocktail (Cat. No. 87786).
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Yes. We offer the ExpiSf Expression System which is highly scalable with a range of protocols available, spanning from 24-deep-well plates to at least 3 L shake flasks. In addition, larger scale growth of ExpiSf9 Cells in ExpiSf CD Medium is possible.
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We recommend using an MOI of 5 for expression studies using ExpiSf Cells. However, when expressing a protein for the first time, you may want to follow the recommendations below to optimize titer of your recombinant protein:
- MOI: Infect cells at varying MOIs (e.g., 1, 2, 5, and 10) and assay for protein expression.
- Baculovirus volume: Infect cells using varying volumes of baculovirus (e.g., 100 µL, 200 µL, 500 µL, 1 mL) and assay for protein expression.
- Time course: Infect cells at a constant MOI or volume, and assay for recombinant protein expression at different time points post-infection (e.g., 24, 48, 72, 96 hours post-infection).
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
An MOI of 5-10 is typically recommended for expression of most proteins with the ExpiSf Expression System. If too much virus is added, unfortunately the cells would die too soon and the protein expression level would go down.
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When ExpiSf9 Cells are efficiently infected, viable cell density will decrease to <80% at Day 3 post-infection. In parallel, cell diameter will increase from 16 µm (uninfected) to 19-20 µm at Day 3 post-infection. Finally, when observed under a microscope, infected cells will have enlarged nuclei and the cytoplasm may contain vacuoles and demonstrate granularity.
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We have observed a decrease in cell infectivity when the cell density is >7 x 10E6 cells/mL at the time of infection. If the cell density is >7 x 10E6 cells/mL 24 hours after addition of ExpiSf Enhancer, it is likely that the initial cell seeding density at Day 1 may have been above 5 x 10E6 cells/mL. We recommend verifying your cell counts using an alternative method, such as trypan blue exclusion using a hemocytometer, to ensure that cells are being accurately counted and seeded for your experiments.
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For the best results, we recommend adding ExpiSf Enhancer at the time of cell seeding, 18-24 hrs prior to virus infection. The enhancer may be added to the flasks without pre-warming.
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One day prior to virus infection, ExpiSf9 Cells should be seeded at a density of 5 x 10E6 viable cells/mL and immediately treated with ExpiSf Enhancer (Day1). On the next day (Day 0; 18-24 hrs after enhancer addition), the cells should be at a density of 5-7 x 10E6 cells/mL and ready for infection. It is important to infect cells within the 18-24 hr period after enhancer addition, for optimal performance.
Note: Incubating ExpiSf Enhancer-treated ExpiSf9 Cells for longer than 24 hrs may result in decreased infection efficiency and low protein yields.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
The ExpiSf Enhancer is designed to work with the ExpiSf Expression System for maximal protein expression. If the enhancer is not added 18-24 hrs prior to infection with baculovirus, there will be a greater than 50% loss in protein expression.
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We recommend using a FACS-based method for quantifying gp64-positive, infected cells, as described in the ExpiSf Expression System User Guide (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017532_ExpiSfExpressionSystem_UG.pdf ). This method allows you to determine the titer of your recombinant baculovirus stock in infectious virus particles (ivp) per mL of culture in less than 24 hours.
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We don't recommend amplifying the baculovirus stock higher than P3, as more defective interfering particles will be produced and a decrease in protein expression level will occur. Notably, we have observed a gradual decrease in baculovirus infectivity at each amplification step; therefore, we recommend avoiding multiple rounds of amplification. Instead, we recommend following our suspension-based transfection protocol using the ExpiFectamine Sf Transfection Reagent so that you can generate large quantities of high-titer, high-quality P0 virus.
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We suggest using a baculovirus stock with a titer of >1 x 10E8 infectious virus particles (ivp)/mL for protein expression studies. Using the recommended suspension-based transfection protocol, the typical range of P0 virus titer that we have typically observed is 1-5 x 10E9 ivp/mL (4 days post-transfection).
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The ExpiSf Expression System allows rapid generation of high-titer baculovirus using a suspension-based transfection protocol. ExpiSf9 Cells should be seeded at a density of 2.5 x 10E6 viable cells/mL just prior to transfection. After seeding, cells are returned to the incubator for 0-30 mins and then transfected following the recommended protocol. If desired, ExpiSf9 Cells can also be transfected in adherent culture format. When using adherent ExpiSf9 cell culture, cells should be seeded at a density of 1 x 10E6 cells/well in 6-well plates just prior to transfection. After seeding, cells are returned to the incubator for 30-60 minutes to allow them to adhere to the plate, and then transfected following the recommended protocol.
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As a quick check, we recommend seeding ExpiSf9 Cells at 0.5 x 10E6 cells/mL in 25 mL of ExpiSf CD Medium in a 125 mL vented, non-baffled shake flask and checking viability and viable cell density on Days 5, 6, and 7 post-seeding. Typically, ExpiSf9 Cells will reach maximal cell density, in the range of 18-20 x 10E6 cells/mL, around Day 7 post-seeding. The cells will continue to show high viability from Day 7 to Day 8, after which there will be a drop in viability.
Note: The final viable cell density value will be dependent upon the method used to count cells. Significant variability can be observed from different counting methods. If cells are exhibiting significantly different growth profiles, optimization of culture conditions should be performed. In such instances, it is typically useful to test multiple different shake speeds simultaneously to determine which speed provides optimal cell growth and then start with this speed for protein expression runs.
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If ExpiSf9 Cells significantly overgrow a density of 10 x 10E6 viable cells/mL during routine subculturing, we recommend passaging the cells down to a lower cell density of 0.5 x 10E6 - 1.0 x 10E6 viable cells/mL to reduce stress on the cells. Subculture cells a couple of times and monitor cell viability and growth kinetics to ensure that the cells have recovered and are healthy (i.e., ≥90% viability and reaching a viable cell density of ≥5 x 10E6 cells/mL 3-4 days post-passaging) before proceeding with experiments. If cells are allowed to overgrow above a density of 10 x 10E6 viable cells/mL on multiple occasions and growth patterns have been affected, we recommend establishing a new culture by thawing a new vial of cells.
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ExpiSf9 Cells should be passaged at least three times post-thaw and be growing within the ranges specified in the product manual, prior to transfection and/or viral infection. The cells should maintain consistent performance for at least 20 passages if maintained in accordance with the cell culture maintenance guidelines in the product manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017532_ExpiSfExpressionSystem_UG.pdf). For optimal performance, we recommend thawing a new vial of cells after 30 passages.
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ExpiSf9 Cells do not reach log phase growth until a cell density of approximately 4 x 10E6 cells/mL. Therefore, the cells should be allowed to attain a density of 5-10 x 10E6 cells/mL before subculturing to help ensure they have reached log phase growth. For all cell manipulations, we recommend simply swirling flasks to resuspend the cells, not shaking or pipetting the cells vigorously to mix, as this can lead to decreased performance, especially just prior to transfection when cells have attained very high densities.
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In instances where ExpiSf9 Cells are thawed and do not start to grow, or attain relatively low densities in culture, one common solution is to verify the culture volume and shake speed according to Table 1 in the ExpiSf Expression System Smart Start Guide (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017562_ExpiSfExpressionSystem_QR.pdf).
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ExpiSf9 cells should exhibit ≥80% viability at 3 days post-thaw. Within 1-2 passages post-thaw, the cells should be growing with a doubling time of approximately 24 hours. When cells are cultured at 0.5 x 10E6 cells/mL, or 1.0 x 10E6 cells/mL, viable cell density should be approximately 5-10 x 10E6 cells/mL within 3 or 4 days, respectively. If cells are not growing within these approximate ranges within 3 passages from thawing, cell culture conditions will require further optimization.
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Upon receipt of ExpiSf9 Cells on dry ice, our recommendation is to either thaw the cells immediately or place the vials in liquid nitrogen (vapor phase) storage for ˜72 hours to allow cells to acclimate until the time of thaw. We do not recommend storing the cells at -80 degrees C. Once thawed and transferred into room temperature medium in a vented, non-baffled shake flask, we recommend incubating the cells in a non-humidified, non-CO2 atmosphere incubator at 27 degrees C on a shaker platform set to 125±5 rpm for a shaker with a 19 mm or 25 mm orbital diameter or 95±5 rpm for a shaker with a 50 mm orbital diameter. Cells should exhibit ≥80% viability at 3 days post-thaw and should reach their normal 24 hour doubling time within 1-2 passages post-thaw.
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We recommend using the Bac-to-Bac Baculovirus Expression System with the ExpiSf Expression System. We have not tested system performance using other baculovirus expression vector systems (BEVS). If using other BEVS, experimental conditions will have to be established empirically.
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Baculovirus stocks can be stored at 4 degrees C protected from light for up to 12 weeks. Alternatively, they can be stored at -80 degrees C or in liquid nitrogen for longer periods. We do not recommend repeated freeze/thaw cycles of your virus stock. Frozen virus should be stored in small aliquots and not re-frozen once thawed. Following these guidelines, baculovirus stocks can be stored without DMSO or cryopreservatives (e.g., FBS or BSA). In instances of unstable virions, 0.1% - 1% BSA may be added to stabilize the virus. Store the virus stocks in polypropylene containers or siliconized glassware to prevent non-specific binding of virus. They should be re-titered periodically if used as inoculates.
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We always recommend titering your baculovirus stock prior to protein expression experiments. A fast and easy-to-follow FACS-based method for baculovirus quantification can be found in the ExpiSf Expression System User Guide (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017532_ExpiSfExpressionSystem_UG.pdf). However, if you choose not to titer your virus stock, we recommend using 250-500 µL of P0 virus stock for protein expression at the 25 mL culture scale (1:100 to 1:50 dilution). For optimal protein expression, we recommend performing a titration of your virus to determine the optimal amount to be used in your infections.
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No. The ExpiSf Expression System is able to achieve high protein yields because of the way the components of the system have been optimized to work together for maximal baculovirus production and protein expression. ExpiSf CD Medium is a chemically defined, yeastolate-free, high-density growth medium specifically matched to ExpiSf Enhancer. ExpiSf CD Medium is essential to support high-density growth of ExpiSf9 Cells and helps enable high-density infections.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.