Lenti-X GoStix Plus FAQs
Lenti‑X GoStix Plus are designed to quickly and accurately quantify the amount of lentivirus in your viral prep, using only 20 µl of packaging cell supernatant. Ten minutes after applying your sample, a test band will appear in the window of the GoStix cassette at an intensity that correlates with the amount of lentivirus applied. The cassette is then scanned using a smartphone camera or equivalent mobile device running the Lenti‑X GoStix App, which will then calculate a titer (i.e., GoStix Value; GV) based on the intensity of the test band relative to a control band.
Applying lentiviral supernatants to Lenti-X GoStix Plus cassettes
Using the Lenti-X GoStix App to analyze the test results
What are the benefits of using Lenti‑X GoStix Plus to measure lentiviral titer?
Lenti‑X GoStix Plus cassettes provide a fast and simple method for obtaining an accurate viral titer. Traditional methods for measuring titer such as qPCR, ELISA, or functional assays can be fairly lengthy and cumbersome, and are therefore not ideal for monitoring your supernatant to identify an optimal harvest time, or for testing multiple samples. Lenti‑X GoStix Plus enable you to obtain a titer in 10 minutes by simply adding 20 µl of supernatant to the GoStix cassette and then scanning the cassette with your smartphone camera after a brief incubation. The app also records the titer so that you can email yourself the result for record keeping. The chart below compares the time required to measure lentivirus titer with Lenti‑X GoStix Plus relative to traditional methods.
Figure 1. Most commonly used methods of lentiviral vector titration and their associated timelines (in hours). Lenti‑X GoStix Plus: a lateral flow-based method for the detection of lentiviral p24 in supernatants. qRT-PCR: quantitation of viral RNA genomes by qRT-PCR. ELISA: measurement of the amount of p24 capsid protein. PCR: detection of integrated DNA proviruses by qPCR. IFU (FACS): determination of the percentage of infected cells via FACS analysis of RFP/GFP positive cells. IFU (Selection): infectious units determined by quantifying the number of drug resistant colonies in transduced populations.
What is p24?
Recombinant lentiviruses generated for research use contain major structural proteins, including envelope proteins, matrix proteins, and virus core proteins (Figure 2). p24 is a structural protein that comprises a majority of the capsid (or shell) of lentiviral particles, and is encoded by the gag gene (along with the nucleocapsid proteins, p6 and p7, and the matrix protein, p17).
Lenti‑X GoStix Plus and methods such as p24 ELISAs are designed to specifically measure the amount of p24 capsid protein present in lentiviral supernatants, because levels of p24 correlate directly with infectious viral titer.
Figure 2. p24 is a major structural component of the lentivirus capsid.
What is a GoStix Value (GV) titer?
The Lenti‑X GoStix App scans the GoStix cassette for the test and control bands, measures the intensity of each band (Figure 3), and then performs a calculation that generates a metric which we refer to as the GoStix Value (GV). The intensity of the test band on the cassette increases with the amount of p24 protein in your lentiviral supernatant, which in turn correlates with increasing infectious units (IFU). The GV is correlated to infectious titer in the same way that ng p24 or genome copies (gc) correlate to infectious titer when using traditional p24 ELISAs or qRT-PCR assays, respectively, to determine titer (Figures 4–6, see subsequent questions). And just like ng p24 or gc, the GV that is best for each experimental application will vary depending on several factors including the target cells, the MOI required, and the lentiviral vector and packaging system used.
Figure 3. The Lenti‑X GoStix App uses a smartphone camera to determine the relative intensities of the test (T) and control (C) bands and uses the ratio to calculate the titer.
How accurately do GV titers correlate to infectious units (IFU)?
Data presented in Figures 4 and 5 (below) demonstrate a strong correlation between GV and IFU across varying concentrations of sample and varying lentiviral packaging systems.
Figure 4. Lenti‑X GoStix Plus-based titers correlate with transduction titers across varying concentrations. Lenti‑X ZsGreen1 lentivirus was prepared using the Lenti‑X Packaging Single Shots lentiviral packaging system. The sample preparation was serially diluted and added to Lenti‑X GoStix Plus cassettes and densitometric analysis was performed. In addition, each dilution was titrated on HT1080 cells to determine actual IFU/ml. The IFU/ml for the unknown samples was calculated using a GV to IFU/ml ratio determined from a reference virus produced using the same packaging system and the same harvest time as the unknown sample. The IFU/ml calculated from the Lenti‑X GoStix Plus was then plotted against the actual IFU/ml.
Figure 5. Lenti‑X GoStix Plus-based titers correlate with transduction titers across varying packaging systems. ZsGreen1 lentivirus was prepared using three different commercially available lentiviral vector and packaging systems. Samples for all preparations were diluted and added to Lenti‑X GoStix Plus cassettes and analyzed. In addition, each dilution was titrated on HT1080 cells to determine actual IFU/ml. The IFU/ml for each unknown sample was calculated using a GV to IFU/ml ratio determined from a reference virus produced using the same packaging system and the same harvest time as the unknown sample.
Where do I download the app?
You can download the Lenti‑X GoStix App from the App Store or Google Play. Search for Lenti‑X GoStix.
Which devices are supported by the Lenti‑X GoStix App?
The app works on all Apple iPhones from 5 onwards using the iOS 9 operating system or newer. Android devices running Android v4.4 and later are also supported.
How do I correlate GV to IFU/ml or ng p24?
It is as simple as doing a side-by-side comparison. Please click here to view data comparing ELISA- and Lenti-X GoStix Plus-based titers.
What factors can impact a GoStix titer?
When using Lenti‑X GoStix Plus (or any other method, e.g., p24 ELISA, or qRT-PCR) to compare titers between different experiments, the following variables must be consistent: lentiviral packaging systems, lentiviral vectors, transfection methods, harvest times, and scanning devices. If any one of these parameters is changed between experiments, we recommend that you re-correlate the GV to IFU.
How can harvest time affect the GoStix Value?
The GoStix Value (GV) is proportional to the amount of p24 protein produced, which in turn correlates with the length of the time to harvest. Therefore, as demonstrated in Figure 6 (below), a longer harvest time will result in a larger GV, assuming that other conditions (e.g., packaging system, transfection method, etc.) are kept constant. The data presented below also demonstrate that the ratio between GV and IFU/ml varies depending on the harvest time, which is why harvest time must be kept constant to enable calculation of IFU/ml from GV, and comparison across experiments.
Figure 6. p24 production and GoStix Value are dependent on harvest time. ZsGreen1 lentivirus preparations were made in duplicate in accordance with the manufacturer’s instructions using Lenti‑X Single Shots and harvested at the times indicated. After harvest, samples were applied to Lenti‑X GoStix Plus cassettes and analyzed for GV. Virus dilutions were also titrated on HT1080 cells to determine IFU/ml for each timepoint.
What happens if I do not wait 10 minutes for the signal to develop?
The test will not give consistent results if the full development time is not observed and you will not be able to accurately compare your result to previously tested samples. A warning within the app will appear if the Skip button is pressed before 10 minutes have elapsed on the timer.
Can Lenti‑X GoStix Plus be used even in the absence of a smartphone?
Absolutely, however, without scanning the intensity of the band the result will be qualitative (yes, my prep is good; or no, my prep is not ready) rather than a quantitative titer.
Figure 7. Using Lenti-X GoStix Plus to obtain a qualitative result. The test band is only visible when you have a useable lentiviral titer, so Lenti-X GoStix Plus can be used qualitatively in the absence of a smartphone scan. In the experiment above, a clear band was generated by a dilution containing ~5 x 105 IFU/ml (as measured by flow cytometry of transduced HT-1080 cells).
Why do I not need to create a standard curve for Lenti‑X GoStix Plus?
For other commonly used titration kits such as p24 ELISAs and pRT-PCR assays, you always need to compare your test sample to a standard curve that you generate. This is not required for Lenti‑X GoStix Plus because the standard curve for each lot of Lenti‑X GoStix Plus is generated at Takara Bio and loaded onto the app when you open it and connect your mobile device to the internet. In addition to saving you time and money, this also ensures maximum accuracy in GV readings between lots and experiments.
Why do I have to enter the lot number of my kit into the app and where is the lot number found?
In order to generate accurate data, the app requires that you enter the lot number because different lots of Lenti‑X GoStix Plus yield slightly different standard curves (similar to an ELISA). The lot number can be entered by scanning the QR code located on the GoStix cassette pouch, or it can be entered manually (Figure 8).
Figure 8. The kit lot number can be input manually or by scanning the QR code on the packet.
Does my mobile device need to be connected to the internet?
When you first use the app, you will need to connect your device to the internet so that the standard curves for available lots of the kit can be downloaded into the app. The app uses the lot-specific standard curve to calculate the GV. When you purchase a new lot of GoStix, make sure to reconnect your device to the internet so that the standard curves for new lots can applied to your app.
How do I know if my titer is outside the range of the GoStix?
For accurate results, it is important that the intensity of the test band is less than that of the control band. The closer this ratio is to 1, the closer the sample is to exceeding the standard curve. If the ratio is close to 1, perform a 1:2 dilution of your sample and apply the dilution to a fresh cassette, then indicate the dilution factor in the app when scanning, as indicated by the green arrow in the screenshot below.
Is any data saved in the app?
Yes. Your results will be saved in the “Results history” in the main menu, provided that you do not delete the app. To avoid losing data, we recommend emailing yourself the data by pressing the Share button at the bottom of each data set page, as indicated by the green arrow in the screenshot below.