I am going to concentrate lentivirus particles using Amicon tubes (
Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-100 membrane). Since they are expensive I wonder is it possible to reuse them? How? would it be safe for cross contamination for different viruses? If I just use always for the same virus? In which condition should I keep them.
I know that it is usuall for protein concentration protocols but I am not sure about virus.
I am not sure it can be used for viruses.
But you can check the cut off and size of your virus and yes it can be use again, just you should added 20% alcohol and keep it.
I was using Vivaspin 20 Sartorius for lentivirus concentration and they work perfectly. I wouldn't advise reusing the tubes.
Yes, indeed these are single use. Depending on input composition (i.e. media containing serum), re-use may not be possible due to clogging of internal structure of membrane so cleaning inefficient.
Yes, these can be used for concentration of viruses or buffer exchange of virus. Use 50 kDa or 100 kDa MilliporeSigma filter type such as Amicon Ultra's device (0.5 mL, 2.0mL, 4mL, or 15 mL) for lentivirus . Keep in mind that not all manufacturer's molecular weight cut-offs are created equal if using other brands.
If input contains serum or protein, centrifugation will take longer than in a buffer without. Centrifuge at 1500xg (4 mL size takes ~ 30 min. in cell culture supernatant containing ~9E4 titer of lentivirus and 10% serum, recovery >93% and was concentrated 100x).
If you were to try to re-use, then keep with same virus as to not x contaminate. Rinse device with 0.1N NaOH followed by centrifugation using Milli-Q water rinse. If needed, a protease treatment can be performed before the final water rinse. After device rinsing, add water to unit to keep the device in a wet condition between uses. If the membrane dries out, the membrane "skin" can crack.
Let me know if you want more details.
Happy spinning!
Hi Janet, Thank you for your suggestions.
After centrifugation the medium also became concentrated together with virus. I centrifuged 12 ml of medium containing virus. There was dark red concentrated medium above the filter and a light pink medium (about 500ul) at the bottom of tube. I have some questions? Where is the virus? top of the filter? and how can have pure virus with no medium?
Your virus is in the retentate (inside filter unit) along with the concentrated media/ serum. At this point, you can buffer exchange (1-3x) with buffer of choice to reduce the media's lower molecular weight proteins (i.e. albumin), but unfortunately some of the higher ones will not be filtered out. Note that filtrate in the bottom (pink medium) is just the media's ph indicator, phenol red.
i will be using amicon ultra filter for the first time for virus conc. ( lentiviral), which will then be used for ipsc generation. Even after reading so much i really cant calculate the MWCO required for my virus filtration. 4 plasmid vector DNA are being used of sizes- 10.7kb, 5.8kb, 12kb and 11.7 kb. Accounting total to some 40.7 kb. can anyone plz help
Use 100 kDa Amicon Ultra 4 (Merck KGaA UFC810024) or tighter 50 kDa (Merck KGaA UFC805024 and spin at 1500xg. Keep in mind that serum proteins will be concentrated too so the 100K is preferred. Spin in 3-5 minute intervals until desired concentration level achieved. Note that the Amicon devices have Ultracel Regenerated Cellulose and other centrifugal device vendors have other membrane types/ratings such as PES. Reach out if you have any quesitons.
thank you very much janet for your help and that too in time. Like you have said about the types of membrane available. So regenerated cellulose should be used or some other type. what does this imply- Spin in 3-5 minute intervals until desired concentration level achieved. which conc. your are talking about... as in virus conc. , like to be concentrated upto how many ul.... ???
Referring volumetric concentration of your virus sample in the upper retained reservoir. You can monitor volume levels (indicator bars printed on device) by start/stopping to view progress and adjust times as needed to desired volumetric ending volume (i.e. 4 mL start to 200 uL ending). Serum in your sample increases processing time.
For membrane types, RC has a tighter structure than PES, even with the same kDa rating and guidance for kDa rating: RC 1-2x smaller or PES 3-5x smaller than retaining particle, etc.
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