Lentivirus Rapid Quantitation Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
This kit can detect 10^10 viral particles/mL from 100 µL of lentiviral supernatant. This kit measure both the FIV and HIV virus particles	Add QuickTiter Solution A to the sample and incubate at 37ºC for 30 minutes	Read the plate with a fluorescence plate reader using a 480/520 nm filter set.

Upstream tips
This kit can detect 10^10 viral particles/mL from 100 µL of lentiviral supernatant. This kit measure both the FIV and HIV virus particles
Protocol tips
Add QuickTiter Solution A to the sample and incubate at 37ºC for 30 minutes
Downstream tips
Read the plate with a fluorescence plate reader using a 480/520 nm filter set.

Publication protocol

The retroviral particles were produced employing the principle of genetic trans complementation used earlier in our laboratory for studying FIV and HIV replication21,22. These assays utilize three individual expression plasmids to produce different parts of the virus, including a packaging construct that expresses the viral gag/pol structural genes (MB22 for FIV and CMVΔR8.2 for HIV-1). These proteins create the retroviral particles which are capable of encapsulating respective retroviral RNAs expressed from the packageable RNA provided by the plasmids, TR394 (FIV) and MB58 (HIV-1). These transfer vectors carry the hygromycin resistance marker gene cassette that allows monitoring of successful retroviral infectious events carried out by the virus particles thus produced. The number of hygromycin resistant (Hygr) colonies obtained thus are directly proportional to the amount of infectious viral particles produced. An envelope expression plasmid (MD.G) based on vesicular stomatitis virus envelope G (VSV-G) was used to pseudotype the two different retroviral particles. The rationale behind pseudotyping different retroviral particles by a common VSV envelope glycoprotein (Env-gp) was that both of these retroviral particles (FIV and HIV) would then be decorated by similar Env-gp.

The three plasmids for constructing each virus (packaging construct + transfer vector + Env-expression construct) were co-transfected into 293T producer cells, which generated the virus particles containing the hygromycin resistance gene cassette. The virus particles were released by the 293T cells into the tissue culture medium used to culture the cells: Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin, streptomycin, and gentamycin antibiotics. These virus particles were then used to monitor their electrical signatures.

The QuickTiter™ Lentivirus Quantitation Kit (Cell Biolabs Inc., San Diego, CA USA) was used to determine the number of FIV and HIV lentiviral particles in the tissue culture media according the manufacturer's directions (Fig. 5). This kit allowed us to measure both the FIV and HIV virus particles using the same assay (based on their nucleic acid content) rather than using two different virus-specific assays that would have had different sensitivity and specificity against each viral species. Briefly, the harvested viral supernatants from the cells were first clarified under a low speed centrifugation step (300 × g for 5 minutes at 4°C) and filtered through a 0.2 micron filter to remove any cellular debris. Next, the mock supernatants without virus or with either FIV or HIV were digested with nucleases to remove any source of nucleic acid released from ruptured cells or virus in the supernatant. The viral particles in the mock or test supernatants were captured using proprietary beads in the kit that allowed the specific capture of lentiviral particles. The beads were washed two times in the wash buffer provided and the virus was denatured on the beads, releasing the RNA from the captured viral particles. The RNA released was then detected using a CyQuant® GR RNA binding dye using the Perkin Elmer VICTOR™ X3 Multilabel Plate Reader using a 485/538 nm filter set. The viral RNA was quantitated against an RNA standard that was provided in the kit for the quantification purposes which gave a linear curve over 1–1000 ng of lentiviral RNA, as shown in Fig. 5.

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Manufacturer protocol

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