Question
Asked 31st May, 2022
Kananbala Patra
Kananbala Patra

How to revive insect cell line SF9?

Hello everyone!
Recently, i have started working with SF9 cell line from thermo (Cat No. B82501) for which i was able to culture and grow the newly received vial in complete Grace's insect media at 27 degree Celsius in a non-humidified incubator as per the instructions (bright field images attached for reference). But i am not able to witness the viable cells after revival of frozen cells from previous batch. Approximately 10*7 cells/ml cells were frozen with 80% complete Grace's insect media, 10% heat inactivated FBS, and 10% DMSO as per instructions. Even though the doubling time is 24-30 hrs, there is slow growth of cells even after 7days.
Seeking expert advice regarding the possible reason for such slow growth of cells and how to enhance the culture conditions.
Thanks
Kanan
Grace
SF9
Insect Cell Line
Insect
Fetal Bovine Serum

Most recent answer

Ionela Litso
University of Crete
Wen Liu is it required to use medium supplemented with 20% FBS when subculturing the culture or we use it only in the first step?
Cite

Top contributors to discussions in this field

Ravi Kant Upadhyay
Ravi Kant Upadhyay
Richard Christison
Richard Christison
Ron Reiserer
Ron Reiserer
Mohamed Rasheed Gadalla
Mohamed Rasheed Gadalla
Archit Garg
Archit Garg

All Answers (5)

Wen Liu
Texas A&M University System Health Science Center
This is pretty common. What I can suggest you is that after the Sf9 cells were seeded on the plate (usually 15-30 min), remove the supernatant media that contains DMSO immediately, then replace with fresh complete Grace's insect containing 20% heat inactivated FBS and then incubate at 27C for 2 -4 days. Check for growth every every day, and then split it into new flasks when Sf9 cells' monolayer growth occupy 90% surface.
Cite
Kananbala Patra
Institute of Life Sciences
Thank you Wen Liu for your response.
I usually do the same after overnight incubation, like if i seed the cells in the evening, change the media next day morning. But yes, will change the media within 30 mins and increase the FBS% as per your suggestion. At the same time, i would like to ask does washing with PBS hamper the cells? We normally do it for mammalian cells, but it is not mentioned anywhere in the user manual for insect cell lines.
Cite
Wen Liu
Texas A&M University System Health Science Center
My experience is that you really do not need to wash the cells with PBS between media changes.
Cite
Kananbala Patra
Institute of Life Sciences
Thank you Wen Liu for sharing your experience.
Cite
Ionela Litso
University of Crete
Wen Liu is it required to use medium supplemented with 20% FBS when subculturing the culture or we use it only in the first step?
Cite

Similar questions and discussions

Unhappy Sf9 cells?
Question
4 answers
  • Asked 14th Mar, 2022
  • Yukino KobayashiYukino Kobayashi
Hello insect cell experts,
I am currently culturing Sf9 insect cells for a protein expression and noticed some tadpole-like cells (red circled in the picture). They seem to reduce/disappear when cells are close to confluence. Are they just unhappy Sf9 cells, or could it be a contamination?
These cells are recovered from a cryo-stock and not yet transfected.
For the culturing, I use supplemented Grace's medium with 10% FBS and Pen/Strep.
I would appreciate if anyone with insect cell experience could tell what they are.
Thanks in advance!
Why are my Sf9 cells not growing?
Question
21 answers
  • Asked 26th Apr, 2013
  • Rachael BaileyRachael Bailey
I purchased a brand new vial of Sf9 cells costing over £200 and thawed them exactly according to the recommendations. However, after 8 days they have still not grown at all - there is some loss of viability however the majority of the cells are still alive. They are cultured in Sf900II media with no serum as advised. Has anyone had a similar problem?
Why do my for my sf9 cells have an higher doubling time?
Question
2 answers
  • Asked 12th Jan, 2016
  • Tang XiaoqiangTang Xiaoqiang
As stated,the doubling time for my sf9 cells were increased:the cells were seeded at the density of 106/ml and three days later,it only reached 2*106/ml with higher than 90% viability.The cells were newly thawed and subcultured for 4-5  passages.The conditions,including temperature(27oC) and shaking speed(120rpm)followed exactly my previous experiment,which gave good results.The same frozen stock were thawed before and they grown very well. Can anyone have any ideas?
Issues with Sf9 cell culture?
Question
28 answers
  • Asked 12th Jun, 2013
  • Rachael BaileyRachael Bailey
I have tried 8 different stocks of cells from 4 different sources. I have tried 2 different types of media - Sf900 and TC100 both with and without serum and also different incubators and growing at room temp. Each time the cells grow fine for a few days and then suddenly disintegrate (see attached pics) - does anyone have any ideas?
How to store insect sf9 cells?
Question
1 answer
  • Asked 18th Jul, 2023
  • Julien LevyJulien Levy
Is it possible to keep them in -80 freezer or should we keep them in liquid nitrogen?
How to increase qantity of protein expression in insect cells?
Question
10 answers
  • Asked 31st May, 2022
  • Philipp CzermakPhilipp Czermak
Hi all!
I was wondering if anyone has some recommendations concerning optimizing the yield of protein expression from insect cells.
Maybe some short information concerning my situation:
I am working with two related proteins that seem to be very sticky and hard to purify. They contain intrinsically disordered domains and have an isoelectric point of about 9-9.5.
I could handle the fact that it is very tedious to purify if the yield was fine. However, the expression levels are very very bad. I am cloning, expressing and purifying proteins for almost 4 years and I have never seen a protein that expresses this bad. It expresses to an extent that resembles the same intensity as the cell lysate background. If I load a whole cell lysate sample on an SDS-PAGE I could not tell what of those bands are my protein since it is indistinguishable from all the other bands.
What I have done so far is infect several 50ml Sf9 cultures with different amounts of P3 virus stock (1:50, 1:100, 1:150, etc.) to determine ideal expression conditions that I check via cell viability, size, density and ultimately a western blot against the protein after 3 and 4 days post-infection. This way I determined the best expression condition that I can come up with so far (96% viability at point of harvest). But, as I said, the amount of protein is so low that it can be compared with impurities in the prep. This expression is therefore not suitable to work with if I don't want to express with 100L of Sf9 culture.
Any suggestions? Might the expression in Hi5 for example be helpful? Maybe they deal a bit better with this type of protein? Might secretion be an option and increase the yield?
Cheers
Sf9 cells float or clump and float after passaging. Is there a clue for that?
Question
9 answers
  • Asked 25th Feb, 2016
  • Nourhan ElsahlyNourhan Elsahly
I am working on sf9 cell line that is maintained on Grace's Insect media. lately the cells started to clump and float after new passages. there are still some cells attached on the flask but almost 50% are floating on the media surface. 
I am culturing SF9 cells for protein expression. Not sure if they are contaminated. I see black dots even after media change or passage. Help?
Question
1 answer
  • Asked 22nd Jun, 2022
  • Soumyadeep MandalSoumyadeep Mandal
Kindly refer to the image attached.
Is the sf9 culture contaminated ?
Question
3 answers
  • Asked 3rd Mar, 2022
  • Isabel LeiteIsabel Leite
HI, I have seen in the cellular environment with sf9 a strange structure, it is similar to a long fiber, sometimes short too. There are not many, it is possible to identify between 3-5 such structures in my flasks. The cells grow normally, viable, there is no contamination turbidity aspect in my culture medium and cottony aspects do not appear in the medium.

Related Publications

Characterisation And Analysis Of Human Interferon-Y Glycoforms Produced In Baculovirus Infected Spodoptera Frugiperda (SF9) And Estigmena Acrea (EA) Cell Lines
Chapter
  • Jan 1995
A large percentage (~50%) of the human therapeutic proteins that are of clinical relevance are glycosylated [1] and these carbohydrate moieties can have a great influence on efficacy, antigenicity, and circulatory half life of the protein. There can be considerable diversity in the carbohydrate structure attached at each glycosylation site. Several...
View
View
EFFECT OF SELECTED INSECTICIDES ON SF9 INSECT CELL LINE
Article
Full-text available
  • Jan 2013
The toxic effect of three insecticides: dimethoate (organophosphate insecticide), acetamiprid (neonicotinoid insecticide) and deltamethrin (pyrethroid insecticide) were evaluated in vitro on cultured Sf9 cell line. Cell growth inhibition was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Regression Analys...
View
Got a technical question?
Get high-quality answers from experts.
Ask a question