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Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
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Sf9 cells
Posted by: Lakshmi Kantham (IP Hidden, New member, 2)
Date: April 25, 2005 12:47PM
Hi,
Recently I started working with Sf9 cells for expression and purification of proteins of human origin in milligram quantities. I received live Sf9 cells from BD Pharmingen in Max XP serum free medium. They tell me the healthy cells attach and form monolayers and the floaters are non-healthy ones. However, my cells seem to attach when I first plate them out. But, they have a tendency to grow in clusters and float even before they get 100% confluent. They seem shiny, round and healthy when I examine byTrypan blue exclusion method. So, I am wondering whether I can keep going - passage, infect and produce viral stocks even though they don' t adhere. There is another problem I am having. I have some old viral stocks I like to test the viability and concentration. I tried to do the EPDA (End point dilution assay) using the above cells. Since they don't stay attached I am having trouble determining the titers. I see some bloated, watery, unhappy looking cells. But, not able to judge the titers properly. And also, I plan to do the suspension cultures in Shaker incubator (where we normally grow our bacterial cultures) using either conical flasks or cell culture storage flasks. I am keeping the lab temperature around 22 degrees and the incubator seems to be holding 27-28 degrees. Is it feasible to do this?? What is a good rotating speed if I do this?? Are there any other inexpensive ways of growing these suspension cultures for viral amplification and protein expression?? I would appreciate suggestions and comments on what is the best way to go about with this. Lakshmi
Re: Sf9 cells
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: April 29, 2005 07:26AM
For suspension cultures of Sf9 cells, 0.1% Pluronic-F68 (Life Technologies) can be added to the medium. To avoid problems due to oxygen limitation during the infection experiment, you may wish to keep the flasks only about half full. Agitation speed can be set from 65-75 rpm. You may also want to read the paper below which suggests that in standard spinner flasks growth is limited by mixing and oxygen transfer. I would contact BD tech support about your problems getting the cells to remain adherent.
Biotechnol. Appl. Biochem. (2003) 38, (15–18) (Printed in Great Britain) Improvements in spinner-flask designs for insect-cell suspension culture Gopinath V. Annathur1, Jennifer L. Pierce, Rodney G. Combs, Anurag S. Rathore, Amit Banerjee and David E. Steinmeyer
Re: Sf9 cells
Posted by: Lakshmi Kantham (IP Hidden, New member, 2)
Date: May 5, 2005 09:44AM
Thank you for the reply.
BD has sent me another batch of Sf9 cells. Interestingly, these cells unlike the last batch seem to attach very firmly to flasks and culture dishes. I am having real trouble detaching them off of the plates for passaging. BD manual doesn't recommend Trypsin/EDTA for detaching cells or using cell scrapers. I tried forcing the medium several times using 10 ml pipette. Some cells come off, most die and they don't look happy when I passage them. So, I have two types of Sf9 cell lines. One non-adherent and grows happily in suspension. The other strongly adherent and hard to detach and passage. Any tips for getting cells off from plates without hurting them and viable for passaging? Lakshmi Kantham
Re: Sf9 cells
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: May 9, 2005 10:09AM
Insect cells attach very tightly to substrates under serum-free conditions and thus can be difficult to detach (passage). First, carefully aspirate off the old media using a sterile pipet. Gently add new media. Then, firmly hit the side of the flask several times to loosen the cells. Follow this by carefully pipetting the medium up and slowly shooting it down the flask over the cells. This can take sometime, but remember to be patient as dislodging the cells with excess force can kill or injure them. Avoid foaming the media or creating lots of air bubbles. Concentrate on one part of the flask until you notice the cells being removed, then move to another area and repeat. i think you may have just be using too much force while attempting to dissociate the cells. good luck!
Sf9 cells seem granulated
Posted by: SW (IP Hidden, New member, 3)
Date: December 29, 2005 07:12AM
hi
i am at passage 4 of my sf9 cells which i grow in suspension, and i notice when i plate them in 6 wells for transfection, the cells are having lots tiny black dots inside, giving a "granular" look. as if they have been infected by i dunno what. can someone tell me what could be the problem here? I have to start transfecting for producing recombinant proteins...and I think my cells are not healthy, but how do I overcome this problem? Thanks
Re: Sf9 cells
Posted by: devi (IP Hidden, New member, 1)
Date: January 12, 2006 07:16PM
Hi !
I am working on the Plant P450s and am using SF9 cells for P450 expression. I have cloned my gene of interest and prepared bacmid using the Bac to BAc manual.Then started with the expression part.I was continously getting P420 peak.I then tried the coexpression of my gene of interest bacmid with reducatse bacmid.I did found P450 peak along with the P420 peak.I was really amazed by these results Now i m trying to repeat the experiment ,but its not working at all.First of all all eukaryotic P450s are suppose to express perfectly without even expressing the reducatse.Second of all its not reproducible.Whats happening, i m really confused.I am struggling with this expt since last year.Please help me.
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