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常见问题

Q
InvivoGen常见问题解答
FAQ about InvivoGen products
物理特性
◎ A. Packaging
QuestionsAnswers
1. Why does the vial I received appear empty?Lyophilized powder may rest in the bottom angle of the vial or near the stopper. Add an appropriate solvent as indicated on the datasheet to dissolve the product for use.

Note that some of our products do appear as clear/ translucent films which can be harder for visual inspection, e.g. MPLA, CRX-527, PMA and LyoVec™.

2. My product has arrived damaged. What should I do? Please report to us with photos of the damage and specify the PO and LOT number.
3. Why is catalogue code printed on the vial label sometimes different from the official one?As some of our catalogue codes had been updated, the old version of cat code may remain on the vial label.
E.g. The vial label of vac-pova shows vac-efova instead. vac-efova was the old product code of vac-pova.
In case of some products, catalogue codes on vial label are our internal production reference, they may differ from the one on offical label.
E.g. vial label: tlrl-nacgas, product label: tlrl-nacga


◎ B. Preparation of Stock Solution
QuestionsAnswers
1. What can I do if my product is not dissolved at the instructed concentration and solvent?In general, customers can warm the solution at 37°C and vortex until complete resuspension. If it still does not re-dissolve,  please contact our technical support for further assistance.
Note that it can be difficult to solubilize lipids like MPLA by vortex. The suspension may appear to contain floating fine particles. Further sonication (e.g. at 25KHz 35°C for 5 mins) can help disperse these particles.


2. Can I use the solvent other than suggested by TDS?We recommend customers to use our instructed solvent to obtain optimal dissolution.
E.g. Conventional R848 is poorly soluble in water and soluble in organic solvents. Yet, InvivoGen's R848 is designed as water soluble, customers can dissolve it using the endotoxin-free water provided.
E.g. Some may use isopropanol to solubilize TDB, yet our internal testing found that it does not dissolve well in isopropanol at 1mg/ml. We recommend customers using DMSO as suggested by TDS .
3. Can I prepare a higher stock concentration than instructed?We recommend customers to prepare the stock solution at instructed concentration.
E.g. Poly (I:C) already forms a thick mixture when dissolving at 1mg/ml. Though heating at 65-70°C for 10mins would mix well, we do not advise a higher stock concentration than 1mg/ml, as it will be more difficult to solubilize Poly (I:C) and may affect the annealing process.


◎ C. Storage and Stability
QuestionsAnswers
1. How long will the product be stable? Where can I find the expiry date of the product?The stability information is stated in TDS. If handled and stored appropriately, the product should be stable up to the expiry date assigned. While the expiry/retest date of the product is indicated on Certificate of Analysis, you can request the CoA from our customer service, specifying the catalogue code, LOT number (and PO number if available).  
Note that for antibody and plasmid products, there are no expiry dates/retest dates indicated.


2. Can the product be stored at a different temperature? The storage temperatures that we indicate on TDS are the optimal temperature for storage. We do not guarantee the stability of the product being stored at a different temperature.
3. If I cannot finish using the product at once, can I store the remaining product?Repeated freeze/thaw the remainings will affect product activity. We recommend preparing the aliquots in advance.
4. Why does the stability of LyoVec™ - complexed ligand products last much shorter after resuspension?Products pre-complexed with transfection reagent will have a shorter shelf life compared to naked products. Upon resuspension, the pre-complexed product is only stable for 1 week at 4 °C. If you would like to extend the stability, please purchase the naked product and transfection reagent separately. Upon resuspension, the naked product is stable for 1 month at 4°C and 1 year at -20°C.


◎ D. Ordering
QuestionsAnswers
1. What is the guideline to bulk discount?There is no fixed order quantity to get a bulk discount. Please contact us to request a quotation for bulk ordering.

◎ E. Documentation
QuestionsAnswers
1. Why is the date of signature in CoA later than expiry date sometimes?This may happen on a re-issued CoA for products with expired retest dates. The date of signature is updated whenever our QA/QC department edits the CoA.
2. Do you have GMP certificates for production?No. We do not offer any GMP certified product. All of our products are strictly for research use only and not for human or veterinary use.
3. Do you have toxicological information about the product?Toxicological data is not available as stated in MSDS Section 11 (MSDS can be found on each product website).
4. Where can I find sterility information?You can refer to TDS or CoA which states the absence of bacterial contamination (e.g. lipoproteins and endotoxins) in our products.


◎ F. Other Technical Questions
QuestionsAnswers
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.

Q
Cosmobio水凝胶常见问题
Cosmobio水凝胶
http://export.cosmobiousa.com/contents/detail.php-product_id=6232.html
物理特性
• 当温度高于37℃,甚至高于60℃时,水凝胶将会发生什么变化?

随温度升高水凝胶将会变硬。

• 在0-60℃温度范围内,溶胶/凝胶/水凝胶发生相态变化的温度范围分别是多少?<br>凝胶相可逆变化的温度范围是多少?

0℃-15℃:溶胶

15℃-20℃:溶胶和凝胶的中间体

20℃:相态变化的温度转折点

20℃以上:水凝胶

37℃以上:随温度升高,水凝胶开始变硬。

Q
Invivogen基因筛选抗生素常见问题
基因筛选抗生素常见问题
物理特性
多种筛选抗生素可以用于同一细胞系吗?
可以的,所有的筛选抗生素均可同时或任意组合使用。
为什么在初始细胞培养的过程中不推荐使用筛选抗生素?
我们建议在细胞传代两次之前不要添加筛选抗生素,因为细胞在初始培养过程中是脆弱的,抗生素可能会降低细胞活力。并且耐药标记的充分表达使细胞获得对这些筛选抗生素的耐药性是非常重要的。
如何停用Invivogen的筛选抗生素?
对于停用Invivogen所有的筛选抗生素(Zeocin™, Hygromycin, G418, Phleomycin, Puromycin, and Blasticidin),建议如下:

● 在培养基中加入5%的NaOH(氢氧化钠)和0.1%的NaClO(次氯酸钠)。

● 在室温放置5-10分钟。

该操作能有效地使培养基中的所有抗生素失效。

可以提供Invivogen筛选抗生素在细胞培养基中的半衰期数据吗?
非常抱歉,Invivogen没有关于筛选抗生素在细胞培养中的半衰期数据。Invivogen在测试时是在新鲜培养,而非储备培养基中添加筛选抗生素的。但是如果有需要的话,可以在培养基中补充筛选抗生素,并储存于4℃,储存时间不超过一周。
Q
Invivogen抗污染抗生素常见问题
抗污染抗生素
物理特性
细胞培养过程中如何避免细胞污染?
1 进行细胞培养时确保处于无菌环境,并使用恰当的灭菌技术。

2 细胞培养过程中所使用的培养基或添加的培养物均要确保无菌无污染。

3 定期使用显微镜或检测试剂盒监测细胞培养基,以确保无微生物污染。

4 使用抗污染抗生素。在原代细胞培养或克隆过程中微生物污染是难以发现的,Invivogen公司为此设计了具有预防功能的防污染试剂。Invivogen公司可提供的抗污染抗生素有Plasmocin™ prophylactic,Normocin™, Primocin™ 及Fungin™。

如何鉴别细胞培养过程中被哪种微生物污染?
如果细胞培养过程中存在细菌和/或真菌污染,可以使用光学显微镜进行鉴别。因为它们的生长速度很快,可以使细胞培养基出现浑浊或菌斑,一般48小时后即可用肉眼检测。随后可使用检测试剂盒对这些微生物种类进行鉴定。然而,支原体污染不能直接用肉眼观察,也不能通过光学显微镜进行鉴别,只能通过特有的分析方法(如支原体检测试剂盒)进行检测。因此,在细胞培养过程中支原体污染很容易被忽视。Invivogen提供的PlasmoTest™支原体污染检测试剂盒结果可靠,分析准确,尤其是可以排除假阳性或不确定的结果。
针对不同微生物的污染,应如何选择Invivogen的抗污染抗生素?


应用

产品名称

支原体

细菌

酵母

真菌

规格

产品货号

预防

Plasmocin™ prophylactic

*



25mg(10×1 ml)

ant-mpp

Normocin™

500 mg(10×1 ml)

ant-nr-1

Primocin™

500 mg(10×1 ml)

ant-pm-1

Fungin™



75 mg(5×1.5 ml vails)

ant-fn-1

检测

PlasmoTest™



1 kit(250 samples)

rep-pt1

清除

Plasmocin™ Treatment

*



500 mg(2×1 ml)

ant-mpt

Plasmocure™

*



100 mg(1 ml)

ant-pc

Normocure™




100 mg(2×1 ml)

ant-noc

Fungin™



75 mg(5×1.5 ml vails)

ant-fn-1


在冻存细胞之前的细胞培养过程中,可以添加抗污染抗生素吗?
可以的,在冻存细胞之前可以添加Plasmocin™ prophylactic(预防支原体污染试剂)或广谱抗生素Normocin™。但是,在这之前需使用光学显微镜鉴定细胞是否被细菌或真菌污染,或使用PlasmoTest™支原体检测试剂盒检测是否存在支原体污染。
Invivogen是否可以提供一种可以同时抵抗细菌、真菌、支原体污染的抗生素吗?
Normocin™和Primocin™这两款抗生素是广谱性的,可同时抵御细菌、真菌、支原体的污染。两者的区别是Primocin™推荐用于原代细胞培养中预防微生物的污染,而Normocin™则推荐用于细胞系培养。
在细胞培养过程中可以同时使用Invivogen的抗污染抗生素及基因筛选抗生素吗?抗污染抗生素会影响基因筛选抗生素的作用效果吗?
在细胞培养过程中可同时使用Invivogen的抗污染抗生素及基因筛选抗生素,抗污染抗生素的使用不会干扰常用的基因筛选抗生素的作用效果,例如G418, Blasticidin, Puromycin, Hygromycin B和 Zeocin™。
Q
Invivogen细胞常见问题
Invivogen细胞常见问题解答
物理特性
收到细胞后,首次进行培养,应该注意些什么?
收到细胞后最重要的步骤是解冻,但亚洲的客户无需进行此步骤,因为Invivogen将细胞运往亚洲地区时使用的是细胞培养板在常温下运输。收到细胞后需要立马进行传代培养,不要放在-80℃或者液氮中,因为这会损伤细胞。


我在HEK和THP1细胞的初始培养过程中有困难,请问有什么建议可以参考吗?
如果您在初始培养细胞的过程中遇到任何问题,请参考以下的建议:

● 对于前2-3次传代的细胞,应置于含20%胎牛血清且无抗生素的培养基中培养。但此条件下培养细胞时间不宜过长,2-3代之后的细胞应使用含10%胎牛血清的培养基进行培养。

● 请定期检查细胞状态,不要使细胞的汇合度达到100%。

● 当冻存细胞时,请继续培养其他未冻存的细胞,以防冻存细胞有问题。


如果你正在培养THP1细胞,以下是额外需要注意的地方:

● 对于THP1细胞,我们建议细胞浓度在3 x 105 -7 x 105 cells/mL时进行传代,一般为5 x 105 cells/mL。如果低于这个浓度,细胞将需要很长时间才能生长,如果高于2 x 106 cells/mL,则可能会产生毒性。

● 在每次传代之间,请不要使用离心机分离细胞。THP1细胞在条件培养基中生长的会更好,因此当传代时,不要弃去上一代培养细胞时所使用的全部培养基,留1-2 mL,并加入新鲜的培养基。但是原来培养基的占比不能超过50%,因为这样会导致细胞无法获得足够的营养去生长。


为什么要在细胞传代2-3次之后才可加入筛选抗生素?

由于最初的耐药标记物水平较低,细胞对筛选抗生素会更加敏感。因此,建议等待2 - 3代后再添加筛选抗生素,以避免在前几代时对细胞造成进一步的应激。


建议用什么血清来培养InvivoGen的细胞?

建议使用无内毒素的血清来培养报告细胞系。InvivoGen在培养细胞时,会要求供应商提供其所使用血清的COA,以确保低水平的内毒素,以免激活NF-kB通路。


细胞培养基中应添加多少浓度的青霉素或链霉素?
建议使用100 U/mL的青霉素和100 µg/mL链霉素。
如果用于细胞培养的培养基中所含的L-glutamine(L-谷氨酰胺)浓度与说明书中不一致会有问题吗?

没有问题的,只要培养基中L-谷氨酰胺的浓度不低于2 mM就可以。大多数DMEM配方中含有4mM的L-谷氨酰胺,但一般情况下培养基中含有2 mM的L-谷氨酰胺即可维持细胞的正常生长。


细胞培养过程中可以使用谷氨酰胺或谷氨酰胺衍生物GlutaMax吗?

谷氨酰胺或谷氨酰胺衍生物GlutaMax均已通过测试,可以使用,在使用过程中与添加L-谷氨酰胺的细胞相比未发现有任何差异。


为什么推荐使用热灭活的血清培养细胞?

许多InvivoGen的细胞系都是通过SEAP(分泌型胚胎碱性磷酸酶)报告系统进行改造的。在很多情况下胎牛血清(FBS)中含有的微量碱性磷酸酶(AP)会干扰检测结果。因此,为了降低背景干扰,建议使用灭活血清培养Invivogen的所有细胞。


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