Resources

Growing insect cells successfully – Part II

In our previous blog we talked about the early stages of growing insect cells when you might just have revived a frozen stock and set up either monolayer or suspension cultures (or both).  If you have established a viable suspension culture – and here we are talking primarily about insect cells such as Sf21 or Sf9 – you will probably monitor them fairly closely for their rate of growth.  The

Growing Insect Cells Successfully

With Autumn, or Fall as some of you may call it, upon us everyone seems to back in action in the lab again and hopefully growing insect cells successfully.  Regular readers of these blogs will know that we frequently return to the question of how to propagate insect cells.  The reason for doing so is because growing insect cells successfully is fundamental to producing recombinant proteins using the baculovirus system.

Baculovirus Transfer Vector Plasmid Compatibility

A frequent customer query concerns baculovirus transfer vector plasmid compatibility with either flashBACTM or other systems for making recombinant viruses.  Obviously, if you are buying flashBACTM and our pOET range of transfer vectors you won’t have a problem.  However, given that baculovirus expression vectors have been around since 1983 and many labs have produced variations around a theme, it is not surprising that confusion can arise. Most baculovirus expression vectors

Pre-GMP Manufacture of Recombinant Proteins

While OET is currently unable to offer GMP manufacture of recombinant proteins or viruses, we can offer a premium service for pre-GMP manufacture.  This can serve as a very useful bridge to full GMP projects conducted by other service providers.  So what is the advantage of a pre-GMP step? It really boils down to cost.  Some of our own R&D is moving in the direction of needing GMP but we

Storing Cells at -80°C?

Every so often we are asked if storing insect cells at -80°C is a viable option if you haven’t got access to liquid nitrogen cryogenic facilities.  Our standard response is that this is not a good idea for long term storage of viable cells.  This is largely based on historical dogma that says that cells do not preserve their viability for long at these temperatures.  However, since one of our

When to Passage Sf9 Cells

An important factor in baculovirus expression is assessing when to passage Sf9 cells, which are commonly used to make recombinant viruses or for protein production.  In our last blog we talked about how to make the transition from monolayer to suspension cultures and how maintain cells in a healthy state during this time. To define what is meant by ‘healthy’ cells,  Figure 1 shows a sample of healthy Sf9 cells

Suspension Sf9 Cells from Monolayer Cultures

The transition to suspension Sf9 cells from monolayer cultures is not difficult but does depend on harvesting the attached cells at the right time. Too early and it is extremely hard to detach the cells from the culture flask. Too late and the cells will be in such poor condition that they may not recover when in suspension. Trying to describe in words how the monolayer culture should look prior

Healthy Cells Over the Holiday

We want to talk about maintaining healthy cells over the holiday period spanning Christmas and New Year.  (Some of our regular readers may recognize most of the following from blogs we have posted in previous years, but we think it is worth repetition for new users of the baculovirus-insect cell system). Most labs, academic and industry, will have some time off over the next few weeks. It is often a

baculoQUANT™ Titration of Recombinant Baculoviruses

There are many methods available for the titration of recombinant baculoviruses.  One of the most convenient is the use of the quantitative polymerase chain reaction (QPCR), which assesses how much virus DNA is present in a freshly-amplified stock.  This is compared with a standard curve derived from a control virus stock to give an infectious titre expressed as Q plaque forming units (pfu) per millilitre.  OET Ltd market the baculoQUANT™

Reviving Cells from Liquid Nitrogen

The process of reviving cells from liquid nitrogen, or indeed from transport on dry ice, can be a little difficult on occasion.  Two events at OET recently have highlighted this. Firstly, we received a new cell line that came with a very complicated procedure for its culture – it is a mammalian species, quite different from your run of the mill HeLa’s and HEK293’s.  Its culture is known to be

Co-infection with Baculovirus Vectors for Multiple Protein Expression

A question that comes up from time to time is the use of co-infection with baculovirus vectors for multiple protein expression in insect cells.  The need arises if you have produced a number of baculovirus vectors that each synthesise a component of a multi-subunit complex, such as an enzyme or a virus like particle.  Intuitively, you know that it is probably necessary to add many virus particles per cell to

Baculovirus Transfer Vectors Compatible with flashBAC™

A question we are asked from time to time is to identify baculovirus transfer vectors compatible with our flashBAC™ system. We obviously nominate our own extensive range that is available but frequently someone has an old plasmid from years ago that they want to reuse. Originally they may have paired it with a linear DNA system such as BaculoGOLD or BacPAK6 or even wild type DNA where recombinant virus selection

Variable Expression of Multi-Component Protein Complexes

Variable expression of multi-component protein complexes is increasingly necessary as more sophisticated projects are attempted.  But how can this be achieved?  Most of our expression work is based on the baculovirus system, in particular our flashBAC™ vectors that permit easy recombinant virus generation in a one-step process.  These insect-specific viruses offer an ideal platform for producing a wide range of recombinant proteins including nuclear, cytoplasmic, membrane-bound and secreted examples. But

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