Lentivirus-LC3 production service for Autophagy Flux Detection

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Adenovirus Adv-GFP-LC3 Autophagy Biosensor Adv-mRFP-GFP-LC3 Autophagy Biosensor

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AAV-LC3 User Manual

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Lentivirus-LC3 User Manual

Autophagy is a highly regulated homeostatic degradative process where cells destroy their own components via the lysosomal machinery and recycle them. This process plays both protective and deleterious roles in many diseases, including Alzheimer’s disease, aging, cancer, infection and Crohn’s disease. Elucidating the correlation between autophagy and apoptotic cell death has become the focus of a great deal of research. Members of the LC3 family play a key role in the maturation of the autophagosome. Lysosomal turnover of the autophagosome marker LC3-II reflects starvation-induced autophagic activity, and detecting LC3 by immunoblotting or immunofluorescence has become a reliable method for monitoring autophagy and autophagy-related processes, including autophagic cell death.

Genemedi has launched series of lentiviral packaging service of autophagy related biosensors, in which GFP and/or RFP tags are fused at the C-termini of the autophagosome marker LC3, allowing to detect the intensity of autophagy flux in real-time with more accuracy, clarity and intuitiveness. These biosensors provide an enhanced dissection of the maturation of the autophagosome to the autolysosome, which capitalizes on the pH difference between the acidic autolysosome and the neutral autophagosome. The acid-sensitive GFP will be degraded in autolysosome whereas the acid-insensitive RFP will not; Therefore, the change from autophagosome to autolysosome can be visualized by imaging the specific loss of the GFP fluorescence, leaving only red fluorescence.

Besides, Genemedi has constructed several other fluorescent biosensors to study the different stages of autophagy flux, making the autophagy study much easier.

1. Pre-packaged, ready-to-use, fluorescently-tagged with monomeric GFP & RFP.

2. Higher efficiency transfection as compared to traditional chemical-based and other non-viral-based transfection methods. Ability to transfect dividing, non-dividing, and difficult-to-transfect cell types, such as primary cells or stem cells.

3. Non-disruptive towards cellular function.

4. Reveals changing cellular conditions in real time. Enables visualization under different cell/disease states in live cell and in vitro analysis.

Our optimized production of lentiviral vector and strict quality control systems supply customers with a high titer of functional recombinant lentiviral vectors. Two methods are employed to determine viral titers: physical titer (VP/mL) and functional titer (TU/mL). Physical titer is calculated by the level of protein, such as p24, or viral nucleic acid. The functional titer, a calculation of the active virus that can infect cells, is much less than the physical titer (100-1000 fold lower). The method we adopted is functional titer, which is an accurate solution for testing virus accurate activity and MOI. The physical titer can only reflect the number of virus particles, but not reflect the true viral activity, which will cause a large error in subsequent infection experiments.

1. For further information about lentivirus administration and transduction, please see Lentivirus User Manual.

2. For further information about autophagy study, please see Lentivirus LC3 Autophagy Flux Detection User Manual.

1. How can I increase the transduction efficiency?

Answer

Try varying particle-to-cell ratio (PPC), incubation volume, temperature and, cell density (if adherent cells are transduced). For adherent cells, we recommend a confluence of about 70%. Following the PPC, adjusting the volume is the next best parameter to change to optimize protein expression.

2. What is the difference between LC3A, LC3B and LC3C, or LC3-I and LC3-II?

Answer

LC3 is a soluble protein with a molecular mass of ∼17 kDa and is distributed ubiquitously in eukaryotes. It is expressed as the splice variants LC3A, LC3B, and LC3C which display unique tissue distribution. All LC3 isoforms undergo post-translational modifications, especially PE conjugation (lipidation) during autophagy. Upon autophagic signal, the cytosolic form of LC3 (LC3-I) is conjugated to PE to form LC3-PE conjugate (LC3-II), which is recruited to the autophagosomal membranes.

3. Can I use the LV-mRFP-GFP-LC3 products to study autophagy in cells in combination with a lentivirus transfection?

Answer

Lentivirus transfection may actually induce autophagy, skewing the results. Make certain to include appropriate controls and make sure to leave an appropriate time (48-72hrs) post transfection to lower the basal autophagy level.

4. Will the GFP signal of mRFP-GFP-LC3 that anchored at autophagosomes and fused with lysosome relight after the cell fixed?

Answer

GFP is no longer delighted reversibly once GFP-LC3 signal was destroyed by the acidic environment.

5. Can transient expression of RFP-GFP-LC3 differ badly from stable expression?

Answer

Transient transfection of GFP-LC3 can leads to the formation of non-autophagic LC3 puncta due to overexpression. Reports shows that GFP-LC3 is aggregate prone protein. We suggest the use of stable cell line for assessment of GFP-LC3 puncta.

6. Which software do you usually use for counting the number of LC3 dots in the cell?

Answer

Image J does work. For LC3 puncta, we suggest using the "Watershed" plugin from Image J. But proper threshold adjustment is critical. CellProfiler is another choice. First use the top hat filter. Then with the Identify Primary Objects module, manually adjust the Intensity threshold to optimally select the punctas of the right intensity.

7. What is a Lentivirus?

Answer

Lentivirus is a subfamily of the retrovirus family. Lentiviruses can deliver significant amounts of genetic information into host cells and integrate it into the cellular genome. Genetically-engineered lentiviruses are therefore used as one of the most efficient tools of gene delivery.

These lentiviruses contain a viral promoter which is used to control the expression of a transgene or shRNA but no virulence genes anymore. Together with several other security modifications this makes them safe to use in the laboratory.

8. How does it work?

Answer

Co-transfection of packaging plasmids and transfer vector into a packaging cell line allows efficient production of lentiviral particles which are released into the cells supernatant.

Viral particles harvested from the cell supernatant can transduce a wide range of both dividing and non-dividing mammalian cell types. Upon infection with lentiviral particles, the single stranded RNA (ssRNA) is reverse-transcribed and the resulting double-stranded DNA (dsDNA) stably integrates into the genome of the host providing for long term transcription of gene of interest or shRNA.

9. How long is chloroquine half life when I treat a cell line for an autophagy study?

Answer

Chloroquine is an attractive drug agent effective for the treatment of not only malaria but also inhibition of autophagy, which is a promising effect for anti-tumor therapy. Half-life time of this drug is approximately 18 hours in vivo due to the degradation by the liver, but the stability of chloroquine in vitro experiments is expected to be longer. It will depend mainly on the cell line your are working on and their metabolic activity. For example HepG2 cells (human hepatocarcinoma cells) have a strong capacity to metabolize drugs. this might not be the case for other cell line of different tissular origin. I would suggest to add chloroquine when you change your growth medium.

10. What medium is best to use to induce LC3 puncta in HeLa cells?

Answer

We recommend the media used by Axe et al. (2008) JCB 182: 685-701. The recipe is 140 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM glucose, and 20 mM Hepes, pH 7.4, Add 1% BSA and pass through a 20um filter before use.

11. What is the best applicable inhibitor of autophagy?

Answer

I prefer to use CQ (chloroquine) in all of my researches about autophagy inhibition. The using of CQ is quite easy, since it is easy dissolve in water (unlike 3-MA in DMSO). One time I have been used 3-MA, actually I did not like to work with it. Bafilomycin is another choice for you.

12. When to add bafilomycin to study autophagy?

Answer

it is better to do a time course to be sure. We use at least two times (2 and 4 hours) to starved the cells and we add baf for 2 hours (for 4 hours: the 2 last hours). We then follow LC3-II and P62 by WB. To see the degradation of p62, sommetime 4 hours is better than 2hours.

13. Which neuronal cell line should I use for autophagy level?

Answer

SHSY5Y and PC12 are two well established neuronal cell lines, human and rat cell lines, respectively. You may also try Neuro2A, it is a mouse cell line.

14. How to distinguish selective and non-selective autophagy?

Answer

You can analyze selective mitophagy by interaction/co-localization study of mitochondrial marker protein and the autophagy specific adaptor protein LC3II.

15. When to Add the autophagy treament in cell culture models?

Answer

First, some cells are very sensitive to these inhibitors and you need to optimize your conditions. The second point, if you like to stop autophagy process after inducing it to compare whether it is a cell death or cell survival you may add it just hours (2-3) before treatment. However, many articles prefer to use it as pre-treatment 1 hour before any other treatment.