Product Description
Advanced BioMatrix offers PhotoHA®, a methacrylated hyaluronic acid (HAMA) for photocrosslinkable hydrogels. These hydrogels provide native-like 3D HA gels with the unique attributes to be prepared at various concentrations and photocrosslinked to provide various gel stiffness. The kit comes with 100 mg of lyophilized methacrylated hyaluronic acid and ruthenium and sodium persulfate photoinitiators (400-450 nm visible light photocrosslinking).
| Item | Catalog Number | Package Size | Storage Temperature |
| Methacrylated Hyaluronic Acid | #5212 | 100 mg | -20°C |
| RutheniumPhotoinitiator | #5246 | 100 mg | Room Temperature |
| Sodium Persulfate Photoinitiator | #5247 | 500 mg | Room Temperature |
Hyaluronic acid is the most abundant glycosaminoglycan in the body being an important component of several tissues throughout the body. While it is abundant in extracellular matrices, hyaluronic acid also contributes to tissue hydrodynamics, movement and proliferation of cells, and participates in a number of cell surface receptor interactions.
For the majority of cell types, it is recommended to add additional ECM proteins to the hyaluronic acid hydrogels. The proteins provide important cell binding sites.
Storage:
The product ships on frozen gel packs. Upon receipt, store the PhotoHA®at -20°C. Store the Ruthenium and Sodium Persulfate at room temperature. The product and components are stable for a minimum of 1 year at receipt in powder form.
Once solubilized, the PhotoHA®can be stored at 2-10°C for 1 month. The photoinitiator can be stored for no more than 2 weeks once solubilized.
| Parameter, Testing, and Method | MethacrylatedHyaluronic Acid#5212 |
| Sterilization Method | Filtration |
| Sterility - USP modified | No growth |
| Form | Lyophilized Powder |
| Package Size | 100 mg |
| Storage Temperature | -20°C |
| Shelf Life | Minimum of 6 months from date of receipt |
| Shelf Life After Reconstitution | 1 Month |
| Degree of Methacrylation | 45-65% |
| Molecular Weight | 100-150 kDa |
NMR Analysis | Characteristic |
Hydrogel Young's Modulus E (Pa) | Characteristic |
Swelling Characteristics of PhotoHA®:
50 μL hydrogels fabricated in 4.7 mm diameter molds were imaged before and after incubation in phosphate buffered saline at 25°C for 24 hours. The diameter of hydrogels were quantified using ImageJ software. Statistical comparisons between groups (n=3) were performed via one-way ANOVA with post hoc testing and significance determined atp < 0.05.
Compressive Modulus of PhotoHA®:
Dynamic mechanical analysis (Q800, TA Instruments) was performed on 50 μL hydrogels fabricated in 4.7 mm diameter molds. Hydrogels were secured within a fluid cup via a 0.01 N pre-load and compressed to 30% strain at a rate of 0.5 N min-1. The Young’s modulus of each hydrogel was calculated as the slope of generated stress-strain curves between 10% and 20% strain. Statistical comparison between MeHA concentrations (n=3) was performed via Students t-test with two-tailed criteria and significance determined atp < 0.05.
Reaction Behaviorof PhotoHA®:
Rheological time sweeps (AR2000 stress controlled rheometer, TA Instruments; 0.5% strain, 1 Hz, 25°C) of MeHA crosslinking with exposure to UV light (=320-390 nm) and in the presence of 0.05 wt% Irgacure 2959 (I2959). After 1 minute, the macromer solution (i.e. MeHA and I2959) was exposed to UV light, resulting in the plateau of moduli before 5 minutes.
Directions for Use
Download the full PDF versionor continue reading below:
3D Hydrogel Preparation:
Note: Employ aseptic practices to maintain the sterility of the product throughout the preparation and handling of the collagen and other solutions.
Recommended concentrations are 5-30 mg/ml(0.5-3.0%).
Note: The following recommended instructions are for a 1% hyaluronic acid (HA) methacrylate solution. Adjustments to this protocol may be required for various concentrations.
- Add 10 ml of 1X phosphate buffer saline (PBS), water or cell culture media to the 100 mg of lyophilized methacrylated HA powder.
- Mix on a shaker table or rotator plate until fully solubilized (~30 to 60 minutes) at 2-10°C.Note: Solubilization times may vary depending on the desired concentration and volume of PBS, water or medium added.
- Calculate the volume of photoinitiator to add by multiplying the volume of solubilized hyaluronic acid by 0.02. If the resulting number is 200 ul, for example, you will add 200 ul of ruthenium and 200 ul of sodium persulfate.
- Solubilize the required amount of ruthenium (per step 3) at a concentration of 37.4 mg/ml in 1X PBS or cell culture media.
- Solubilize the required amount of sodium persulfate (per step 3) at a concentration of 119 mg/ml in 1X PBS or cell culture media.
- Add the ruthenium to the hyaluronic acid solution and fully mix until solution is homogeneous.
- Add the sodium persulfate to the hyaluronic acid/ruthenium solution and mix until solution is homogeneous.
- Add your cells to the hyaluronic acid/photoinitiator solution.
- Dispense your hyaluronic acid/photoinitiator/cell solution into the desired dish (ie. 6-well plate, 48-well plate).
- For photocrosslinking, place solution directly under a 400-450 nm visible light crosslinking source.
Product Q & A
Yes. There are quite a few publications citing various hyaluronidase protocols for digesting PhotoHA hydrogels. Here are a few:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5460858/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4840832/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986518/
The HA we usecomes frombiotechnological production, where the hyaluronic acid is extracted from the cell walls of the bacteria Streptococcus zooepidemicus.
Product Cell Assay
Human mesenchymal stem cells (20 x 106 / mL) were encapsulated in 50 μL hydrogels (~ 4.7 mm x 2 mm). Hydrogels (1 wt% PhotoHA®) were fabricated with 0.05 wt% Irgacure 2959 and exposure to 2 mW/cm2 light (320-390 nm) for 10 minutes. After 24 hours, encapsulated cells were stained with calcein AM and ethidium homodimer and subsequently imaged on a Leica SP5 confocal microscope (using FITC/TRITC sequential scans).
Product Applications
PhotoHA® Methacrylated Hyaluronic acid can be used to form hydrogels for ex-vivo engineering of autologous cartilage tissue[1] or as a mesenchymal stem cell carrier in cartilage repair[2].
Because the stiffness can be widely adjusted by altering concentration or UV-light exposure, methacrylated HA has been used to measure the effects of matrix stiffness on cell phenotype and function[3][4].
Methacrylated HA can be used for 3D bioprinting (extrusion[5], inkjet[5] and photolithographic[6]) to create structures that promote osteogenic differentiation of MSC’s[7].
The high tunability of hyaluronic acid methacrylate allows it to be mixed with, and reinforce other types of hydrogels (such as collagen, or gelatin methacrylate)[8].
References:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5717235/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627486/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5541838/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5447944/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5615317/
https://www.ncbi.nlm.nih.gov/pubmed/21773726/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5460858/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5748291/
Product References
References for PhotoHA®:
Khetan, Sudhir, and Owen Corey. "Maintenance of stem cell viability and differentiation potential following cryopreservation within 3-dimensional hyaluronic acid hydrogels."Cryobiology(2019).
Poldervaart, M. T.et al.3D bioprinting of methacrylated hyaluronic acid (MeHA) hydrogel with intrinsic osteogenicity.Plos One12,(2017).
Product Certificate of Analysis
Safety and Documentation
Safety Data Sheet
Certificate of Origin
Product Disclaimer
This product is for R&D use only and is not intended for human or other uses. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices.
美国AdvancedBioMatrix（简称ABM） www.advancedbiomatrix.comAdvancedBioMatrix（简称ABM）是美国一家著名的生物公司，获得了AllerganInc的授权(Allergan用25年时间不断完善胶原蛋白相关的产品的生产工艺)，将Allergan的专业和技术用于蛋白生产与检测，致力于为组织工程、细胞分析及细胞增殖等研究领域提供优质稳定的产品。AdvancedBioMatrix不断丰富已有产品线，目前可为三维细胞培养提供各种胶原蛋白、纤连蛋白、玻连蛋白、水性凝胶、不同粘度与分子量的透明质酸以及低代成纤维细胞等。在美国全部产品授权Sigma销售。AdvancedBioMatrix是组织培养，细胞分析和细胞增殖三维（3D）应用的生命科学领域的领导者。我们的产品被公认为纯度，功能性和一致性的标准。我们在生产，分离，纯化，冷冻干燥，细胞培养和蛋白质测试，粘附肽，附着因子，底物刚性和其他3D矩阵产品方面拥有丰富的专业知识。我们的专业技术和知识正在被用来确保我们的产品质量最高，批次之间一致且易于为我们的研究客户使用。
美国AdvancedBioMatrix是3D组织培养、细胞检测和细胞增殖等领域实验解决方案的佼佼者。AdvancedBioMatrix在分离、纯化、冻干、细胞培养和蛋白检测、多肽粘附、附着因子、基质硬度和其他3Dmatrix 产品开发方面有着丰富的经验。AdvancedBioMatrix的研发经验和专业知识确保其产品可达到最佳质量，并保证产品之间一致性，方便研究客户使用。以下为AdvancedBioMatrix3DMatrices 产品竞争优势：1. 提供高纯度和成分确定的胞外基质；2. 超过1000余篇文献引用PureCol产品，品质非常均一；3. 在3D培养基领域可提供最全面的产品线；4. 唯一可提供特异性刚性有机硅基板的公司（CytoSoft）;5. 唯一可提供可溶性丝纤蛋白的供应商（可运用于多种3D培养）；6. 如果客户首次接触3D胶原凝胶，AdvancedBioMatrix还是唯一的预制胶原蛋白(PureColEZGel)供应商；
以下产品为AdvancedBioMatrix全球畅销品：1.PureCol 牛源I型胶原蛋白 3mg/ml#5005-100ML2.Nutragen牛源I型胶原蛋白 6mg/ml#5010-50ML3.FibriCol 牛源I型胶原蛋白 10mg/ml#5133-20ML4.VitroCol 人源I型胶原蛋白 #5007-20ML5. 弹性蛋白原 #5052-1MG6.ECMSelectArraykitUltra-36#5170-1EA7.CytoSoft（刚性可变的基底，AdvancedBioMatrix最新添加产品5190-7EA）8. 人III型胶原蛋白 #5021-10MG9. 人IV型胶原蛋白 #5022-5MG10.SilkFibroin溶液 #5154-20ML11.Fibronectin#5080-5MG12.Vitronectin#5051-0.1MG
ebiomall.com
>
>
>
>
>
>
>
>
>
>
>
>
1）做免疫组化的片子最好不要同时做HE染色，因为那样会掩盖阳性着色的结果；而且用苏木素复染细胞核也是淡染，若要在免疫组化的片子上观察形态学的改变，除非有非常典型的表现，一般是有很大的难度的。
2）你是培养的细胞做免疫组化吗？如果是的话，那就每个样本只有一张片子吧？唯一的办法是进行免疫双染，同时做CD34和AFP（甲胎蛋白）。我觉得后者阳性便可以或多或少的获得向肝细胞分化的证据。
3）用HE染色来识别造血干细胞向肝细胞分化，也就是说要看到肝细胞的典型形态学改变后才可以下结论。问题是：早期的肝细胞仅凭形细胞态学观察并不能很容易的被识别。病理大夫看组织切片，重要的是看其组织学结构，对肝细胞也是这样。如果单拿出来一个细胞，还是培养的细胞，染色后真的不容易区分。所以我觉得你设计的用HE染色来确定早期细胞向哪个方向分化不是很有说服力。
4）如果是切片，也就是说每个样本可以有多张切片，那可以在连续切片上分别染CD34和AFP，拍照时选择相同视野，也有一定说服力。但是还是不如双染法更可靠。
5）用双染最大的问题在于：这两者都表达在胞浆中，染色容易相互覆盖。所以在双染的方法选择上你还要再考虑一下，可不可以用免疫荧光双染？这样也很有说服力的
GlucosestarvationcausestranslocationofAMPKβ2tothelysosomeinHEK-293cellsthatisdependentonN-myristoylation.Theexperimentwasperformedinβ2KOcellsasinFig.1c,exceptthatthelysosomalMarkerLAMP1(taggedwithRFP)wasco-expressedwiththewild-typeormutantAMPKβ2.Upperpanelsshowmergedimagesstainedblue(4′,6-diamidino-2-phenylindole(DAPI),nuclei),red(LAMP1,lysosomes)andgreen(AMPKβ2,detectedusingantibodyvalidatedine),incellsincubatedwithorwithoutglucosefor20 min.Lowersmallpanelsaremagnificationsoftheareasindicatedbydashedboxesintheupperpanels,showing(LtoR)redandgreenchannelsandmergedimages.
下面的这段话是图注，图注的意思我明白，但是我想知道merge后的图看什么颜色的荧光，蓝色是细胞核，红色是lysosome(位于胞质），绿色是AMPKβ2，该实验是想观察AMPKβ2是否转位到lysosome上了，如果确实发生了AMPKβ2转位到lysosome上，那么merge后是红色与绿色融合在一起，是吗？融合在一起发什么颜色的光了？
（2）另一方面，使抗原抗体结合更稳定。一般不需要,但对表达较弱的抗原可能有用,4度和37度时分子运动方式不同,前者分子碰撞机率和运动速度小于后者,后者结合更快,但敏感性也提高了并易造成非特异染色。
（3）其实，我更赞同后一种说法，因为我尝试把肝脏或睾丸片子从4度过夜拿出后，直接用PBS洗没发生过脱片现象。事实胜于雄辩！
有没有朋友使用碧云天的细胞衰老β-半乳糖苷酶染色试剂盒做过细胞衰老，本人小白，求相关细胞处理的过程及数据分析方法。贴壁细胞六孔板需要接种多少，染色后怎样计数进行数据处理。谢谢啦！
石蜡切片组织茜素红钙染色试剂盒产品说明书（中文版）
主要用途
石蜡切片组织茜素红（ALIZARINREDS）钙染色试剂是一种旨在使用标准化的化学分离石蜡方法和鳌和技术，使钙离子和茜素红S产生复合物，来分析存档中的石蜡包埋的组织切片中橘红色钙沉积现象的权威而经典的技术方法。该技术经过精心改良、成功实验证明的。主要适用于石蜡包埋组织切片的钙沉积和钙化结节检测。广泛用于骨细胞或组织病理生理的研究。产品严格无菌，即到即用，操作简捷，性能稳定，显色清晰。
技术背景
茜素红（ALIZARINREDS）是一种蒽醌（anthraquinone）衍生物：9,10-二氢-3,4-二羟基-9,10-二氧代-2-蒽磺酸单钠盐（9,10-Dihydro-3,4-dihydroxy-9,10-dioxo-2-anthracenesulfonicacidsodiumsalt），又称媒介红3（MordantRed3）或茜素磺酸钠（Sodiumalizarinesulfonate）。其分子式为C14H7NaO7S，分子量为342.25。茜素红和钙离子以鳌和方式形成复合物，用以识别组织细胞的钙盐成分。钙盐变化是骨细胞增殖分化和骨组织成骨潜能的标志之一。通过茜素红染色，产生桔红色沉积，但会受到其它金属元素的干扰。
产品内容
脱蜡液（ReagentA）自备
补水液A（ReagentB）100毫升
补水液B（ReagentC）100毫升
补水液C（ReagentD）100毫升
补水液D（ReagentE）100毫升
清理液（ReagentF）30毫升
染色液（ReagentG）10毫升
产品说明书1份
保存方式
保存在4℃冰箱里；染色液（ReagentG），避免光照；试剂具有腐蚀性，注意操作安全。有效保证3月
用户自备
小型染色缸：用于石蜡切片的脱蜡操作
光学显微镜：用于组织细胞染色后观察分析
实验步骤
一、脱蜡处理
1．取出10片待测的石蜡包埋的组织切片（注意：石蜡包埋前，组织切片须用10％甲醛4℃条件下，固定16小时，此步很重要）
2．（选择步骤）放进80℃烘箱，孵育30分钟
3．（选择步骤）室温下静置15分钟
4．按下表依次放进小染色缸里孵育
染色缸
孵育时间
50毫升脱蜡液（ReagentA）
15分钟
50毫升脱蜡液（ReagentA）
15分钟
50毫升脱蜡液（ReagentA）
15分钟
50毫升补水液A（ReagentB）
3分钟
50毫升补水液B（ReagentC）
3分钟
50毫升补水液C（ReagentD）
3分钟
50毫升补水液D（ReagentE）
3分钟
5．小心移去切片上的补水液D（ReagentE）
6．小心加上200微升清理液（ReagentF）在切片上，铺满整个切片样品表面
7．室温下孵育5分钟
8．小心移去切片上的清理液（ReagentF）
二、样本染色处理
1．小心加上200微升染色液（ReagentG），铺满整个切片样品表面
2．室温下孵育2分钟（注意：可以延长至20分钟）；或直至可见桔红色
3．小心移去切片上的染色液（ReagentG）
4．空气中晾干
三、样本澄清处理
1．小心加上200微升补水液B（ReagentC）在切片上，铺满整个切片样品表面
2．小心移去切片上的补水液B（ReagentC）
3．重复实验步骤1和2二次
4．小心加上200微升补水液A（ReagentB）在切片上，铺满整个切片样品表面
5．小心移去切片上的补水液A（ReagentB）
6．重复实验步骤4和5二次
7．小心加上200微升脱蜡液（ReagentA）在切片上，铺满整个切片样品表面
8．小心移去切片上的脱蜡液（ReagentA）
9．重复实验步骤7和8一次
10．放上盖玻片或封片
11．即刻在一般光学显微镜下观察：钙沉积阳性细胞呈现桔红色
注意事项
1．本产品为50次操作
2．操作时，须带手套
3．试剂具有腐蚀性，注意操作安全
4．石蜡包埋前，组织样品须用10％甲醛4℃条件下，固定16小时，否则造成样品支离破碎
5．所有操作在室温下进行
6．试剂溶液在样品表面时，避免有气泡存在，同时确保铺满样品表面
7．样品染色避免过度，肉眼可见桔红色即可终止染色
8．组织细胞染色完成后，即刻进行光学显微镜观察
9．本公司提供系列组织细胞金属元素染色试剂产品
质量标准
1．本产品经鉴定性能稳定
2．本产品经鉴定显色清晰
暂无品牌问答
