VSV.G‐pseudotyped third‐generation self‐inactivating (SIN) LV were produced by calcium phosphate transient transfection into 293T cells, or by LV packaging or producer cell lines. 293T cells were transfected with a solution containing a mix of the selected LV genome transfer plasmid, the packaging plasmids pMDLg/pRRE and pCMV.REV, pMD2.G or pBA‐AcMNPV‐gp64 (Schauber et al, 2004 ) and pAdVantage (Promega), as previously described (Cantore et al, 2015 ). Medium was changed 14–16 h after transfection and supernatant was collected 30 h after medium change. Alternatively, LV production was induced when LV producer or packaging cells were in a sub‐confluent state, by replacing the culture medium with medium containing doxycycline (Sigma) 1 μg/ml and supernatant was collected 3 days after induction. LV‐containing supernatants were passed through a 0.22‐μm filter (Millipore) and, when needed, transferred into sterile poliallomer tubes (Beckman) and centrifuged at 20,000 g for 120 min at 20°C (Beckman Optima XL‐100K Ultracentrifuge). LV pellet was dissolved in the appropriate volume of PBS to allow 500–1,000× concentration. LV purification from large‐scale (6,000 ml) production was performed as described (Biffi et al, 2013 ; Cantore et al, 2015 ).
Sterile poliallomer tubes
Manufactured by Beckman Coulter
Sterile poliallomer tubes are laboratory equipment designed for sample collection, storage, and transportation. They are made of poliallomer, a type of plastic material. These tubes are pre-sterilized to ensure a clean and safe environment for the samples.
Automatically generated - may contain errors
2 protocols using sterile poliallomer tubes
1
Lentiviral Vector Production and Purification
2
Large-Scale Production of Purified LV
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the NHP study, we used large-scale puri ed CD47hi and MHCfree LV batches, produced by MolMed S.p.A. (now AGC Biologics), on 24 L scale of supernatant, as previously described 15, 17 and formulated in PBS 0.2% human serum albumin. Results of selected quality control assays performed on these batches are reported in Supplemental Table 1. For all the other experiments in vitro and with mice, we used labgrade LV. Lab-grade third-generation self-inactivating (SIN) LV were produced by calcium phosphate transient transfection into 293T cells. 293T cells were transfected with a solution containing a mix of the selected LV genome transfer plasmid, the packaging plasmids pMDLg/pRRE and pCMV.REV, pMD2.VSV-G and pAdvantage, as previously described 17 . Medium was changed 14-16 hours after transfection and supernatant was collected 30 hours after medium change. LV-containing supernatants were sterilized through a 0.22μm lter (Millipore) and, when needed, transferred into sterile poliallomer tubes (Beckman) and centrifuged at 20,000 g for 120 min at 20° C (Beckman Optima XL-100K Ultracentrifuge). LV pellet was dissolved in the appropriate volume of PBS to allow 500-1000X concentration.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!