Q: I cannot detect any gene expression?

Were the cells used for test expression in good condition using a virus with a known titre (by plaque-assay or qPCR on fresh virus)? Has the virus been stored for some time before use - and if so did you add serum or glycerol to maintain the titre? If not, re-titrate your virus and try again. It is often useful to include a control virus as an experimental control, did you include a control virus, and if so did that work? Did you try optimising expression? In particular, sometimes T. ni cells yield protein when Sf9 does not. Very occasionally, the gene is not stable, you can check that the gene is actually in the virus genome by PCR. This can be achieved by harvesting the cellular/virus DNA and use for PCR analysis. it is important to check if the gene is properly under control of the polyhedrin gene promoter (is the first ATG the ATG of your gene) and if you are using tags to detect the gene, check they are in frame. Finally, if you have exhausted all avenues, there are very, very few genes that for unknown reasons do not express. Most have been found to be toxic to the cell. But check all of the above before thinking about this! Here at OET we commonly work on a number of difficult to express proteins, so if you need further information or use our services to help purify your protein please feel free to contact us at info@oetltd.com

Question 1

I am interested in flashBAC™ but would like to know some more information?

Accepted Answer

flashBAC™, which is based on the Autographa californica nucleopolyhedrovirus (AcMNPV), is a one-step baculovirus based protein expression platform that enables fast and simultaneous production of recombinant viruses using a process call co-transfection with insect cells. flashBAC™ is a streamlined process which is quicker and simpler then rival systems that generates a P0 virus stock in only 5 days. We offer an in-depth lab guide that provides further information on flashBAC and our related products, however you can always contact us at info@oetltd.com for more information.

Question 2

Why are my cells not growing well?

Accepted Answer

The most likely problem with cells occurs when they have been allowed to reach the stationary phase before passaging.  If this ‘stress’ happens to a culture 2 or 3 times, then the cells no longer grow properly.  Always check cells on a regular basis and do not let cultures overgrow. If this happens, go back to liquid nitrogen stocks and set up a new culture.  Far more important than the passage number of the cell, is the number of times the culture has been stressed! Cells that are not growing well should never be used to make recombinant viruses, amplify virus, or test for protein production because each of these techniques requires the virus to infect and replicate inside cells and it can only do this is the cells are actively replicating – i.e. in log phase of growth.

Question 3

My virus has not amplified to high titre?

Accepted Answer

High cell density and not being in the log phase will be the most likely problem.  Did you infect cells with low MOI (0.1 pfu/cell)?  High MOI will lead to lower titres and very low MOI will work but you may need to leave the cells longer to achieve high titres. Did the cells look infected? We often look for grainy and swollen nuclei under the microscope with cells showing an often sausage-like shape and with a cell viability of <65% (in Sf9 cells).  Another possible issue is the foreign gene product could be affecting budded virus production?

Question 4

I cannot detect any gene expression?

Accepted Answer

Were the cells used for test expression in good condition using a virus with a known titre (by plaque-assay or qPCR on fresh virus)? Has the virus been stored for some time before use - and if so did you add serum or glycerol to maintain the titre?  If not, re-titrate your virus and try again. It is often useful to include a control virus as an experimental control,  did you include a control virus, and if so did that work?  Did you try optimising expression? In particular, sometimes T. ni cells yield protein when Sf9 does not.

Very occasionally, the gene is not stable, you can check that the gene is actually in the virus genome by PCR.  This can be achieved by harvesting the cellular/virus DNA and use for PCR analysis. it is important to check if the gene is properly under control of the polyhedrin gene promoter (is the first ATG the ATG of your gene) and if you are using tags to detect the gene, check they are in frame.

Finally, if you have exhausted all avenues, there are very, very few genes that for unknown reasons do not express.  Most have been found to be toxic to the cell.  But check all of the above before thinking about this!

Here at OET we commonly work on a number of difficult to express proteins, so if you need further information or use our services to help purify your protein please feel free to contact us at info@oetltd.com

Question 5

We're seeing a ring of dead cells accumulating around the side of our flasks in the shaker and It looks like sludge and accumulates over time. This is happening with Sf9 cells and Hi5 cells, and with different serum-free media. What could be causing this, and how can we resolve it?

Accepted Answer

This phenomenon is sometimes referred to as the "ring of death." While the exact cause is not fully understood, here are some insights and potential solutions:
	1. What is the "ring of death"? 
It's an accumulation of dead cells around the edges of culture flasks.
Often seen in new cell cultures set up from liquid nitrogen storage.
Usually disappears after a passage or two.
Cells, if dislodged, are often in clumps.

2. What might cause this issue? 
It can indicate that the cells are stressed or unhappy with their environment.
Transitioning cells from monolayer to suspension culture can be a factor, especially for Sf9 cells.
Seeding density might play a role; cells seeded too thinly may aggregate in rings.
The type of media used can influence cell health and behavior.

3. How can we address this problem? 
Revive fresh cells from nitrogen storage to mitigate effects of long-term passage.
Maintain cells in suspension culture if possible, rather than frequently transitioning from monolayer.
Ensure proper seeding density (e.g., 0.7 million cells/ml for Sf9 cells).
Consider using a reliable media such as Expression Systems 921 for routine cell growth.
Use consistent flask types (e.g., Corning flasks) to minimize variables.

4.Are there any long-term preventive measures? 
Limit subculturing to no more than 50 times after reviving a batch, even though passage number may not be strictly meaningful for Sf9 cells.
Regularly monitor cell health and growth rates.
Maintain consistent culture conditions and avoid frequent changes in media or flask types.

5. What if the problem persists? 
If issues continue despite these measures, it may be necessary to troubleshoot other factors such as incubator conditions, shaker speed, or potential contamination.
Consider consulting with a cell culture specialist or contacting our technical support team for further assistance.

Remember, occasional "go-slow" periods can occur with Sf9 cells for no apparent reason, which might contribute to the ring formation. If you continue to experience issues, please don't hesitate to reach out to our technical support team for more personalized assistance.

Frequently asked questions

I am interested in flashBAC™ but would like to know some more information?

Why are my cells not growing well?

My virus has not amplified to high titre?

I cannot detect any gene expression?

We're seeing a ring of dead cells accumulating around the side of our flasks in the shaker and It looks like sludge and accumulates over time. This is happening with Sf9 cells and Hi5 cells, and with different serum-free media. What could be causing this, and how can we resolve it?

Expert in Baculovirus protein expression.