Lentiviral vectors based on Human Immunodeficiency Virus-1 (HIV-1) are widely used tools for gene transfer research. These vectors can efficiently integrate exogenous genes or exogenous shRNA (short hairpin RNA) into the host chromosome, thereby achieving long-term stable expression of the target sequence. In terms of infectivity, lentiviral vectors can effectively transduce various cell types, including neuronal cells, hepatocytes, cardiomyocytes, tumor cells, endothelial cells, and stem cells. For cells that are difficult to transfect—such as primary cells, stem cells, and undifferentiated cells—using lentiviral vectors significantly improves the transduction efficiency of target genes or target shRNA. Additionally, it greatly increases the probability of target genes or target shRNA integrating into the host cell genome, enabling convenient and rapid long-term, stable expression of the target sequence. In both in vitro and in vivo research, lentiviruses have become one of the most common vector systems for expressing exogenous genes or exogenous shRNA, and their applications are expanding increasingly.
Lentivirus Concentration Solution (5×) is a polymer-based reagent optimized for lentivirus enrichment. It provides a simple, rapid, and efficient method for concentrating lentiviral particles. The protocol involves mixing lentiviral supernatant with the Lentivirus Concentration Solution at the recommended ratio, incubating for a short period, and then centrifuging using a standard centrifuge to obtain precipitated lentiviral particles. In contrast, other viral concentration methods such as ultrafiltration and ultracentrifugation are time-consuming and cumbersome to operate. They also often suffer from contamination by cell debris and serum proteins, resulting in low viral purity. When using this product for viral concentration, the viral titer can be increased by 10–100 fold, with a maximum viral recovery rate of approximately 90%. This method minimizes viral loss while preserving the biological activity of lentiviruses during the concentration process. The entire experimental procedure can be completed quickly within 4 hours, and excellent recovery efficiency is achieved without the need for ultracentrifugation.
Product Features:
⁕ Simple & Rapid – Easy to operate, no ultracentrifugation required, ensures viral activity, and the experiment takes no more than 4 hours.
⁕ Excellent Concentration Effect – Viral titer can be concentrated by 10–100 folds or more (Transduction Units/mL).
⁕ High Recovery Efficiency – Lentivirus recovery efficiency is as high as 90% or more.
⁕ Wide Application Range – Suitable for all types of lentiviral particles.
Summary of Experimental Procedure:
Figure 1. Flowchart of Lentivirus Concentration Experiment
Experimental Steps:
1. Transfer the culture medium containing lentiviral particles to a sterile container, and centrifuge at 800×g for 10 minutes to remove cell debris.
⁕ To ensure viral activity, freshly collected viral lentiviral supernatant is recommended for concentration.
2. Filter the supernatant through a 0.45 μm filter into a sterile container.
⁕ For membrane filtration, low protein-binding cellulose acetate or polyethersulfone (PES) membranes are recommended; nitrocellulose (NC) membranes are not recommended.
3. Add 1 volume of Lentivirus Concentration Solution (5×) to every 4 volumes of lentivirus-containing supernatant to dilute the Lentivirus Concentration Solution (5×) to the working concentration (1×).
4. Incubate the mixture slowly in a shaker at 4°C for 3 hours or overnight.
⁕ Lentiviral particles remain stable at 4°C for up to 4 days.
5. Centrifuge the mixture at 4000×g for 25 minutes at 4°C. After centrifugation, lentiviral particles may appear as a beige or white precipitate at the bottom of the container.
6. Carefully discard the supernatant, then centrifuge at 4000×g for 5 minutes at 4°C. Discard any remaining liquid completely, taking care to avoid aspirating the precipitate, which contains the lentiviral particles.
7. Resuspend the viral particles in pre-chilled lentivirus preservation solution at 1/10 to 1/100 of the original volume (volume of the initial supernatant). Gently pipette to resuspend the lentiviral particles.
⁕ When resuspending the viral precipitate, pipette gently. Lentivirus can be resuspended in Lentivirus Stable Storage Solution (L1505998), complete medium, or FBS.
8. Aliquot the lentivirus into small volumes and store in a -80°C freezer or liquid nitrogen. Retain a small amount of virus for titer determination.
⁕ Avoid repeated freeze-thaw cycles of lentivirus, as this will reduce viral titer.
Scope of Application:
Suitable for the concentration method of any lentiviral particles.
Precautions:
1. For your health and safety, please operate in a standardized manner and wear a lab coat and gloves when conducting experiments.
2. All lentivirus operations must be performed in a BSL-2 (Biosafety Level 2) biological safety cabinet.
3. This product is for scientific research use only and shall not be used for clinical diagnosis or treatment.
Experimental Case Analysis:
1. Culture cells in a 10 cm cell culture dish; when the cell confluency reaches approximately 75%, start lentivirus packaging.
2. Use the Lentiviral Packaging Kit (L1455942) for lentivirus packaging (the positive control plasmid contains the GFP green fluorescent protein gene). Follow the instructions for specific packaging steps.
3. Prepare target cells: Seed the target cells (293T cells to be infected) into a 96-well plate at a density of (1–3)×10⁴ cells/well, and lentiviral infection can be performed the next day.
4. After 48 hours of virus packaging, collect approximately 9 mL of lentiviral supernatant. Centrifuge at 800×g for 10 minutes to remove cell debris, then filter the supernatant through a 0.45 μm filter into a sterile container.
5. Take 1 mL of the viral supernatant as *Control 1 (pre-concentration control)**. Add 2 mL of this product to the remaining 8 mL of viral supernatant for lentivirus concentration. Follow the instructions for specific lentivirus concentration steps. Retain 1 mL of the supernatant that should be discarded after concentration as Control 2# (post-concentration discarded supernatant control). Gently resuspend the lentiviral particles in 800 μL of Lentivirus Stable Storage Solution (L1505998) to obtain lentivirus concentrated 10-fold.
6. Lentiviral infection of target cells: Aspirate the supernatant from the cells in the 96-well plate. To each well, add 100 μL of fresh medium, 0.2 μL of Polybrene (P1501912), and 100 μL of either concentrated lentivirus, Control 1* (pre-concentration lentiviral stock), or Control 2# (post-concentration discarded lentiviral supernatant).
7. Replace with fresh complete medium 16 hours after viral infection. After continuing to culture the cells for 48 hours, observe the green fluorescent intensity using a fluorescence microscope. The experimental results are shown in Figure 2.
In addition, we verified the lentivirus concentration effect in a variety of common cell lines, and the experimental results are shown in Figure 3.
Figure 2. Observation of GFP expression in target 293T cells infected with lentivirus. (Left) 10× concentrated lentivirus; (Middle) Control 1* (pre-concentration lentiviral stock); (Right) Control 2# (post-concentration lentiviral supernatant to be discarded)
Figure 3. Lentivirus was packaged using the Lentiviral Packaging Kit (L1455942) (a high-titer lentivirus packaging kit). After collecting the lentivirus, it was concentrated 10-fold with the Lentivirus Concentration Solution (5×) (S1506239). The concentrated lentivirus was then used to infect target cells, and the expression level of fluorescent protein was detected. (Left) 10× concentrated lentivirus; (Middle) Control 1: Pre-concentration lentiviral stock; (Right) Control 2: Post-concentration lentiviral supernatant to be discarded
