Get Pure High-Titer Virus with Every Packaging Prep with LentiFuge™

Most experiments that rely on lentiviral technology to deliver genetic elements benefit greatly by starting with pure high-titer virus. With some easy-to-transduce cell systems grown in routine medium, such as DMEM, it may be possible to squeak by with a dilute viral supernatant containing proteins and factors from the packaging cell culture. However, protocols involving cells that require different media (esp. cells that grow in suspension) or cells that are difficult-to-transduce (stem cells, blood cells, etc.) will likely require viral concentrations 100-fold more dense than just the supernatant. Freezing small, highly-concentrated aliquots is much more efficient also.

The problem, of course, has always been how to concentrate the virus without concentrating the exogeneous proteins and other factors present in the supernatant. The traditional approach usually involves either an inconvenient and time-consuming sucrose-gradient ultracentrifugation or a messy PEG precipitation followed by centrifugation which co-precipitates most of the proteins and other impurities in the cells and media.

Fortunately, our experience over the years manipulating lentiviruses has lead to a solution — LentiFuge™ Viral Concentration Reagent. It aggregates viral particles together so they can be isolated and separated from other impurities in the medium by standard high-speed centrifugation. The whole LentiFuge isolation protocol takes just 2 hours. One hour incubation with the Reagent, and a second hour for centrifugation in a Beckman JA-14, JA-10, or similar rotor. After these two short steps, the virus is simply resuspended and either used immediately or aliquoted and frozen.

Data from a recent experiment (below) shows that LentiFuge not only concentrates the virus by 2-fold, but it also removes impurities from the viral preparation. The figure shows the transduction efficiency ratio of concentrated virus versus pre-concentrated virus. With LentiFuge, essentially all the virus was recovered and the LentiFuge Reagent gave much better yields than the more traditional PEG protocol.

Viral supernatant was collected at 48-hrs post-transfection concentrated using either 1) high-speed centrifugation, 2) LentiFuge treatment with centrifugation, 3) PEG (polyethylene glycol) treatment with centrifugation, or 4) not concentrated. The resulting concentrated virus was used to transduce cells in culture.

LentiFuge concentration results in a higher yield and virus purity compared with conventional methods

For more information or to purchase LentiFuge, click here.

Please email with any comments.

Also in Cellecta Blog & News

Perturb-Seq Screening: Cell-by-Cell Analysis of Gene Perturbations Induced by Pooled CRISPR sgRNA Libraries

Read More
Gene Expression Profiling of Single-Cell Samples: DriverMap Targeted Expression Profiling vs SMART Technology

Single-cell expression analysis provides insights about gene expression and cell heterogeneity at the single-cell level. It enables the elucidation of intracellular gene regulatory networks and intracellular pathways that would otherwise be masked in bulk analysis (Massaia et al., 2018). The DriverMap™ Targeted Gene Expression Profiling (TXP) assay combines highly multiplexed RT-PCR amplification with the depth and precision of Next-Generation Sequencing (NGS) to quantitatively measure gene expression of up to 19,000 target genes in a single assay–even down to the single-cell level.
Read More
Comparing DNA vs. RNA Samples for Immune Repertoire Profiling

Adaptive immunity relies on B and T cells that recognize foreign antigens via hypervariable B cell and T cell receptors (BCRs and TCRs). Diversity among B cell and T cell receptors is primarily produced by V(D)J recombination, which involves the shuffling and joining of the variable (V), diversity (D), joining (J), and constant region (C) gene segments. This results in a diverse repertoire called the adaptive immune repertoire (AIR) that comprises multiple individual clonotypes (sequence) for particular receptor chains.
Read More