Hello, I am a master's student currently conducting lentivirus transduction experiments in Jurkat cells. The virus is not effectively entering the Jurkat cells, so I am planning to use an ultracentrifuge for virus concentration. To serve as a cushion, I need to prepare a 10% sucrose solution. Can I use D-PBS for this purpose? Additionally, the protocol I referred to uses Corning #21-040CM, and I have compared it with the composition of wlgene #LB001-02, which my lab uses (add files). There are differences in KH2PO4, NaCl, and Na2HPO4. Is it still okay to use?
Thank you for reading !
Genetic Expert Here! Sorry for the wall of highly technical text, but you're a PhD so I hope you don't mind
Took a large mass of notes, and tried to conglomerate them as coherently as possible, I have trouble explaining my highly technical ideas in a way which is conversational and relax, so I hope you understand.
trouble with lentivirus not effectively entering your Jurkat cells. I think concentrating the virus using an ultracentrifuge is a smart move.
For the cushion in this process, you had said that you planning to use a 10% sucrose solution.
using D-PBS for this is totally a good choice because it helps maintain the right pH and osmolarity, key to keeping your virus intact during concentration. To prepare it, just dissolve 10 grams of sucrose in 90 milliliters of D-PBS, stir it until it’s completely dissolved, and then top it up to 100 milliliters with more D-PBS. Be careful with this. Use your better judgment and don't destroy your samples! I doubt you will, but it's very sensitive as you know
Don’t forget to sterilize it by passing it through micron filter. I'd do 0.22 , and I think concentrating the virus using an ultracentrifuge is a smart move. For the cushion in this process, you’re planning to use a 10% sucrose solution. Now, using D-PBS for this is totally a good choice because it helps maintain the right pH and osmolarity, which is key to keeping your virus intact during concentration. To prepare it, just dissolve 10 grams of sucrose in 90 milliliters of D-PBS, stir it until it’s completely dissolved, and then top it up to 100 milliliters with more D-PBS. Don’t forget to sterilize it by passing it through a 0.22-micron filter heavily with heat or ultrasonic bath! Honestly, I always would go with ultrasonic bath but I know not everybody has those available.
If D-PBS isn’t available, you can also use HEPES-buffered saline or Tris-buffered saline. Regarding the buffer compositions you’re comparing (for reference, I wrote down: Corning #21-040CM and wlgene #LB001-02),
I noticed the differences in KH2PO4, NaCl, and Na2HPO4 too. They can be negligible, or they can be very big depending on your skill level and process. Our individual processes can affect these things, so in this whole instructions I've given you, try to be aware that your personal habits in your genetic processes, affect these time frames and values, and you need to factor that in with your own skill level knowing yourself.
The differences can tweak the osmolarity and pH a bit, but as long as they’re within acceptable ranges, your virus should remain stable. , you should be fine using wlgene #LB001-02, just double-check those concentrations to make sure they don’t stray too far from the recommended levels for lentivirus work.
For your ultracentrifugation, you might want to look into options like Beckman Coulter Optima XPN, Thermo Scientific Sorvall WX+, or Eppendorf Centrifuge 5920 R. These are all solid choices and you can get them from suppliers like Thermo Fisher Scientific, Beckman Coulter, or Eppendorf. You can find these items online easily
then when you’re ready to concentrate the virus,
You can filter your virus-containing supernatant and put it over the 10% sucrose cushion in the ultracentrifuge tube thing. After centrifuging at 25,000 or 26,000 (I don't know which is better) rpm for 2 ish hours at around 4°C, remove the supernatant and resuspend the virus pellet in D-PBS. You have to be really careful when removing it. Use like your studiest hands for this part
For the transduction, seed your Jurkat cells and add the concentrated virus. Adding polybrene can help boost the transduction efficiency. Incubate the cells with the virus, replace the medium, and let the cells incubate for another 48-72 hours before checking the transduction efficiency.
D-PBS for your 10% sucrose solution is definitely fine. The slight differences in buffer compositions should not pose any significant issues as long as you keep the pH and osmolarity in check. Follow these steps, and you should see better virus entry into your Jurkat cells. Does this sound good to you?
Bo-Ram Park
Alex Wolf Thank you so much for explaining the experimental procedure in detail. I was having difficulty since no one around me has tried this experiment before, and your help has been invaluable!
I will plan my experiment again based on the information you provided. Have a great day ;)
Bo-Ram Park please do me the service of how this method works in proliferating the virus for your important medical studies. I would like to know, if you do future studies how this method worked out and the efficiency rate.
I'm confident enough to say it holds up, should work very well. Lines up with the standards for this sort of process genetically, and I have experience researching and or being educated on these areas.
You can feel free to credit , but you don't need to.
Good luck!