| Questions |
Answers |
| 1. Do you have any half life data of your products? |
No, we do not perform half-life testing. |
| 2. Do you have in vivo data for ligands products? Can I use them in vivo? |
We do not have in vivo testing in house. We suggest performing literature search as a reference first. |
| 3. Why can’t I find the molecular weight for some of your products? |
It is possible that the molecular weight of some products cannot be determined accurately due to their intrinsic structure or insolubility.
E.g. PGN is a vast polymer consisting of numerous monomers. We do not know how many monomers make up our purified PGN.
E.g. The nature of LPS causes it to form aggregates of unpredictable sizes (an estimated M.W. could be about 50 kDa). |
| 4. What is the difference between Poly (I:C) HMW and Poly (I:C) LMW? Any tips to choose between them? |
The primary difference is their molecular weight. Poly(I:C) HMW is 1.5-8 kb while Poly(I:C) LMW is 0.2-1 kb in size. Also, the efficiency of TLR3 activation by Poly(I:C) HMW was significantly higher than that by Poly(I:C) LMW, as tested by our 293/TLR3 and Ramos-Blue cells. For details visit https://www.invivogen.com/sites/default/files/invivogen/old/docs/Poly_I_C_LMW_v4-invivogen.pdf
Poly (I:C) HMW is therefore recommended if customers are targeting TLR3 activation; for customers studying RIG-I pathway, they can consider choosing Poly (I:C) LMW. |
| 5. Do you have the exact molecular weight for Poly (I:C)? |
The molecular weight of Poly (I:C) (also Poly(dA:dT) and Poly(dG:dC)) can vary from batch to batch. The lot-specific molecular weight is available to check upon request. |
| 6. What is the advantage of your Poly(I:C) over Sigma’s? |
A study has shown that InvivoGen poly(I:C) exhibited negligible endotoxin level and lower inter-batch variability over Sigma Poly(I:C). For details visit https://doi.org/10.1016/j.bbi.2019.08.006 |
| 7. Why is it necessary to transfect Poly (I:C) for activating RIG-I/MDA5 but not for TLR3? |
Naked Poly (I:C) can be internalized into cells by endocytosis to activate the endosomal TLR3; RIG-I and MDA5 are cytosolic sensors, Poly (I:C) being transfected can reach the cytosol for activation. |
| 8. I can’t find the purity percentage value of LPS products on the website. Do Ultrapure products have a higher purity than Standard products? |
The two versions of our LPS differ in terms of functional purity. Standard version stimulates both TLR2 and TLR4 as it contains contaminating lipoproteins that non-specifically activate TLR2. Ultrapure stimulates only TLR4, having successive steps of enzymatic hydrolysis to ensure no TLR2 activity. |
| 9. Do you provide the endotoxin concentration for LPS products? |
Ultrapure LPS product has the specific endotoxin level (EU/mg) measured for each lot by LAL assay (the data can be found on CoA).
For Standard LPS products, one EU approximately equals one ng of endotoxin, e.g. 5 mg/ml of Standard LPS-EB solution corresponds to 5 x 10⁶ EU/ml. |
| 10. What is the difference between your LPS products? Any tips for choosing the LPS? |
LPS products differ from species to species, primarily in the O-antigen hydrophilic sugars’ length, sequence and self-linkages.
LPS may work differently on cell types based on their strains. As a reference, the most described LPS in the literature is from O111:B4, LPS-EB is therefore a popular choice. |
| 11. What is the difference between CpG-A, CpG-B and CpG-C? |
They are different based on their structural characteristics and activity on human peripheral blood mononuclear cells.
CpG-A ODNs induce high IFN- α production from pDC but are weak stimulators of NF-kB and pro-inflammatory cytokine production.
CpG-B ODNs stimulate strong B cells and NF-kB activation but are weak activators of type I IFN secretion.
CpG-C ODNs combine properties of both class A and C, strongly stimulating B cells and type I IFN secretion.
For details visit https://www.invivogen.com/cpg-odns-classes |
| 12. Is it necessary to transfect flagellin for NLRC4 inflammasome activation? |
We recommend transfecting flagellin for NLRC4 activation, however, this is cell-type dependent. If the cells stably overexpress NLRC4 (e.g. our THP1-NLRC4 Cells), it is possible that flagellin without the transfection agent can activate NLRC4, otherwise, it will require a very high concentration of flagellin to be sufficiently delivered into cytosol for stimulation. |
| 13. Does flagellin-induced NLRC4 activation require LPS priming? |
It is common to have LPS priming before flagellin stimulation. LPS is used for pro-IL-1β, pro-Caspase-1 and NLRP3 induction, and these proteins will help in the NLRC4 inflammasome activation. |
| 14. Is transfection of cGAMP needed for stimulation? |
InvivoGen does not transfect the on-shelf cGAMP for stimulating the reporter cell lines. However, as cGAMP has a poor membrane permeability. You can use a lipid-based transfection reagent to facilitate cGAMP’s delivery to cells for stimulation. |
| 15. Do you provide IC50 and/or EC50 value for the product? |
You may find the values on TDS or validation datasheet for some of our products. |
| 16. Where can I find the sequence of primer that your plasmids used? |
The information is available on our website: https://www.invivogen.com/sequencing-primers |
| 17. The pVITRO1-MCS plasmids carry two elongation factor 1 alpha (EF-1α) promoters, from rat and mouse origins combined to the CMV and SV40 enhancers respectively. Will both promoters display activity in human cells? And will the rat EF1 promoter display activity in mouse cells? |
As EF-1α promoters can be expressed in mammalian cells, they will display activity in human cells, and the rat EF1 promoter will also display activity in mouse cells. |
| 18. Can a human promoter work in mice? |
Our composite promoters allow for strong expression in nearly any mammalian cell line. In fact, matching the species of origin for the promoter to the organism it is being used in (in vitro or in vivo) does not seem to have much of an impact on gene expression levels. The activity of a promoter may vary between cell types, but in general a mammalian promoter should work in any other mammalian model. In our experience promoters from mammalian origin tend to work similarly between human and mouse cells. A good example is for the IFN-beta promoter used in the majority of our HEK-Blue-TLR reporter cells. This is the mouse IFN-beta promoter, however the stimulation of SEAP following TLR induction works perfectly well in the human HEK293 cell line. |
| 19. Do you have a dual promoter vector with fusion proteins tags for each? |
We do not have a dual promoter plasmid with two fusions, but a strategy would be to subclone from our other tagged plasmids. For example while our pVIVO allows for dual expression, it does not allow for GFP fusion. Further, while pDUO allows for dual expression, it does not include GFP at all (either as a fusion or marker).
Only our pSELECT-Tag plasmid allows for fusion to GFP. That plasmid only allows for insertion of one gene. A strategy to express two genes with 1 fused to GFP would be to first clone onto pSELECT-Tag and then subclone onto pVITRO or a similar dual expression plasmid. |
| 20. How can I avoid light interference when plating QUANTI-Luc™? |
QUANTI-Luc™ is light-sensitive. The light interference can result in great variation within a single plate. Recommended steps we take to avoid light interference: wrap QLC in foil/paper, turn off hood lights, work fast, cover plate while walking to reader. |
| 21. What is the difference between QUANTI-Blue™ and QUANTI-Luc™? |
Both are secretory so there is no need to lyse cells, and the choice really depends on your research need.
You may refer to the following information for selection:
QUANTI-Blue™ QUANTI-Luc™
Detection: 630-655nm OD vs Luminescence
Detection apparatus: Spectrophotomer vs Luminometer
Sensitivity: Lower vs Higher
Signal stability: Higher vs Lower |
| 22. Can QUANTI-Luc™ be used to detect Firefly Luciferase? Can it detect luciferases dually? |
QUANTI-Luc™ does not detect Firefly Luciferase. It detects Lucia and Renilla Luciferase. |
