| Description | The Immortalized Adult Mouse Dorsolateral Prostate Cells (INK4 -/- DLP) were derived from a subline of the INK4a mouse, a transgenic knockout that lacks p16INK4a and p19ARF. Both p16INK4a and p19ARF are specific inhibitors of cyclin-dependent kinases Cdk4 and Cdk6 that regulate cell cycle progression. The cells were isolated from the dorsolateral prostate (DLP), which has simple cuboidal epithelium that is somewhat folded. The luminal secretory cells in the epithelium secrete a homogenous eosinophilic substance into the lumen of the DLP. This cell line is a useful model for studying the prostate stroma-epithelium signalling way, which plays an important role in prostate development and cancer progression. |
|---|---|
| SKU | T0655 |
| Species | Mouse (M. musculus) |
| Tissue/Organ/Organ System | Prostate |
| Growth Properties | Adherent |
| Cell Morphology | Fibroblast-like |
| Immortalization Method | Isolated from p16INK4a and p19ARF Knockout Transgenic Mice |
| Applications | For Research Use Only |
| Unit quantity | 1x106 cells / 1.0 ml |
| Cell Type | Immortalized Cells |
| Propagation Requirements | Use of PriCoatTM T25 Flasks (G299) or Applied Cell Extracellular Matrix (G422) is required for cell adhesion to the culture vessels. Grow cells in ECM-coated culture vessels unless otherwise specified in the Propagation Requirements below. The base medium for this cell line is Prigrow IV medium available at abm, Cat. No. TM004. To make the complete growth medium, add the following components to the base medium: fetal bovine serum (TM999)* to a final concentration of 10%, Penicillin/Streptomycin Solution (G255) to a final concentration of 1%, gentamicin to a final concentration of 50 µg/ml, ITS to a final concentration of 1%, L-glutamine (G275) to a final concentration of 2 mM, and DHT to a final concentration of 10-8 M.Change media every 2-3 days.Carbon dioxide (CO2): 5%, Temperature: 37.0°C.* Do not use heat-inactivated FBS for cell culture unless specified otherwise. |
| Disclaimer | 1. For for-profit organizations and corporations, please contact [email protected] for pricing of this item. 2. Sale of this item is subjected to the completion of a Material Transfer Agreement (MTA) by the purchasing individual/institution for each order. If you have any questions regarding this, please contact us at [email protected]. 3. All test parameters provided in the CoA are conducted using abm"s standardized culture system and procedures. The stated values may vary under the end-user"s culture conditions. Please verify that the product is suitable for your studies by referencing published papers or ordering RNA (0.5 μg, Cat.# C207, $450.00) or cell lysate (100 μg, Cat.# C206, $600.00) to perform preliminary experiments, or alternatively use our Gene Expression Assay Service (Cat# C138). All sales are final. 4. We recommend live cell shipments for ease of cell transfer and this option can be requested at the time of ordering. Please note that the end-user will need to evaluate the feasibility of live cell shipment by taking into account the final destination"s temperature variation and its geographical location. In addition, we thoroughly test our cell lines for freeze-thaw recovery. If frozen cells were received and not recovered in your lab under the exact, specified conditions (using recommended culture vessel, media, additional supplements, and atmospheric conditions), a live cell replacement is possible at a cost (plus shipping). 5. All of abm"s cell biology products are for research use ONLY and NOT for therapeutic/diagnostic applications. abm is not liable for any repercussions arising from the use of its cell biology product(s) in therapeutic/diagnostic application(s). Please contact a technical service representative for more information. 6. abm makes no warranties or representations as to the accuracy of the information on this site. Citations from literature and provided for informational purposes only. abm does not warrant that such information has been shown to be accurate. 7. abm warrants that cell lines shall be viable upon initiation of culture for a period of thirty (30) days after shipment and that they shall meet the specifications on the applicable abm Material Product Information sheet, certificate of analysis, and/or catalog description. Such thirty (30) day period is referred to herein as the "Warranty Period." |
| Depositor | Wisconsin Alumni Research Foundation |
- Important Considerations for Immortalized Cells
- Immortalized Cell Handling Instructions Upon Arrival
- Subculturing Protocol
- Freezing Protocol
- Hepatocyte Instructions
- Thawing Protocol
- Immortalized Cell Line Flyer
- abm Cell Line Catalog
- Immortalized Cell Lines Flyer
- Immortalized Cells FAQ
| I want to make sure these cells express my gene of interest before I decide to buy the cell line. Can you provide a sample so this can be tested? | |
We do not carry out downstream characterization or gene expression profiling of our cell lines. To facilitate your preliminary experiments we can provide an RNA extraction (0.5ug total RNA) or cell lysate (100ug/100ul provided in 62.5mM Tris‐HCl, 2% SDS, 10% Glycerol, 50mM DTT, 0.01% w/v Bromophenol Blue) for any of our immortalized cell lines for a small fee. Please inquire directly for more information. The lead time will be around 2 weeks from the time of placing an order (if the item is in stock). | |
| How often do I need to change the media? | |
The media should be changed every 2-3 days. | |
| Why do these cells need bio safety level II? | |
In order to be more cautious, we follow the CDC-NIH recommendations that all mammalian sourced products should be handled at the Biological Safety Level 2 to minimize exposure of potentially infectious products. This information can be found in "Biosafety in Microbiological and Biomedical Laboratories" (1999). Your institution"s Safety Officer or Technical Services will be able to make the call as to whether BioSafety Level I is possible with these cells at your site if required. | |
| Do you sell ECM coated T75 flasks? | |
Yes we can provide a coating service. Please inquire with [email protected] | |
| Is it necessary that we have to use the recommended T25 ECM-coated flasks for growing the cells? Can we use normal ECM coated 60mm/90mm petri plates for growing the cells? | |
We strongly recommend using G299 T25 flasks to ensure recovery of these cells before testing other plates. | |
| What can I coat a larger dish to subculture? | |
We also offer applied extracellular matrix (collagen type I) in liquid form, for the coating of larger flasks and other required plasticware: http://www.abmgood.com/Applied-Cell-Extracellular-Matrix-G422.html | |
| How long can I store frozen vials for? | |
Cells that are properly frozen using an effective cryoprotective agent can be stored in liquid nitrogen indefinitely without affecting their recovery. | |
| When are cells plated for live cell shipments? | |
1 day prior to shipping | |
| Should the cap of the flask be changed before starting the cell culturing step? | |
No, there is no need in sterile biosafety cabinets unless it has contacted any non-sterile condition (e.g. touching the contaminated tip, etc.). | |
| What is the recommended storage temperature? | |
In general, if you received:Live cells: acclimatize for 3-4 hrs at at the recommended conditions stated for the cell line under the propagation section, and then change media afterwards.Frozen cells: Immediately place cells in liquid nitrogen; -180C. | |
| How is cell density crucial for drug selection? | |
If antibiotic selection is applicable to the target cells, we suggest getting rid of all the background cells so that the cell density is kept lower (even 20-30%). However, once the clones are selected by clonal dilution, we don"t need the drug to still be present. If needed, the cell density should be towards the higher end since cells are already selected. Any primary cells still present will be depleted as a result of senescence and the cell population that remains will be resistant to the specific antibiotic. | |
| My cells are not detaching, what method do you recommend to trypsinize the cells? | |
1. Incubate the coated plate containing trypsin solution at recommended temperature indicated in the propagation section for 3-5 min till the cells round up, monitoring from time to time under microscope.2. Diluting G422 (1:1) with PBS and coating for lesser time. Sometimes the collagen content in G422 is higher and thus make stronger bonding with cells.3. You can try reducing the incubation time as well for coating the plate to make a thinner layer. | |
| Why is it important to determine the optimal seeding density? | |
The seeding density we recommend is for when cells are plated to a new vessel. The optimal seeding density should allow cells to attach to the surface and have room to proliferate.If you seed too little, cells may not attach well to the surface (for adherent cells). Seeding density is important as many cells (adherent or suspension cells) need to be in close proximity for better growth. Cell-cell interactions allow cells to communicate with each other in response to changes in their microenvironment. This ability to send and receive signals is essential for the survival of the cell. In other cases, if the seeding density is too low, cells may attach but a retardation in cell growth is observed.If you seed too high, the cells will attach but there is insufficient room for further proliferation and they will stop replicating. | |
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虽然没有具体用过检测这个抗原的抗体，但是如果希望抗体特异性好点，能检测丰度低的蛋白，还是选Abcam吧，Santa有时候全凭人品了，内参抗体什么的还能凑活，其他的比较勉强。
GAPDH
万能内参
主要看你的目标蛋白的分子量多大，“目标蛋白要与内参蛋白的分子量差异大！”
方便检测
核蛋白，有很多种，分子量不同
膜蛋白，有很多很多种，分子量不同
浆蛋白，有成千上万种，分子量不同。
具体实验，具体目的蛋白，具体的内参。
因为内参有好几种，分子量差别也比较大，更换分子量与检测蛋白差距更大的内参就可以避免这个问题
另外也可以先用一抗孵育显色和检测，再用Strip缓冲液洗掉膜上的抗体，重新进行内参的抗体孵育显色检测。这样也可以将检测蛋白和内参显色在同一张膜上
内参抗体：
Beta-Actin mAb (1C7) 细胞总蛋白 42 kD Abbkine A01010 小鼠源单克隆
Beta-Actin mAb (1C7) , HRP 细胞总蛋白 42 kD Abbkine A01015 小鼠源单克隆,HRP偶联
GAPDH mAb (2B5) 细胞总蛋白 36 kD Abbkine A01020 小鼠源单克隆
GAPDH mAb (2B5) , HRP 细胞总蛋白 36 kD Abbkine A01025 小鼠源单克隆,HRP偶联
Beta-Tubuline mAb (3G6) 细胞总蛋白 55 kD Abbkine A01030 小鼠源单克隆
Plant Actin mAb (3T3) 植物细胞总蛋白 42 kD Abbkine A01050 小鼠源单克隆
PCNA mAb (1D7) 细胞核蛋白 28 kD Abbkine A01040 小鼠源单克隆
COX IV mAb (14Y2) 细胞线粒体总蛋白 16 kD Abbkine A01060 小鼠源单克隆
Histone H3 mAb (2D10) 细胞核蛋白 18 kD Abbkine A01070 小鼠源单克隆
标签抗体（Tag Antibody）可用于检测各种商品化表达载体上的标签序列（如：Myc、Flag、His、GST、HA等），籍以分析目的蛋白的表达含量及其功能；
标签抗体：
Anti-Biotin Antibodies
Anti-Dye Antibodies
Anti-FITC Antibodies
Anti-Fluorescent Protein Antibodies
Anti-HRP Antibodies
Beta Galatosidase Antibodies
FLAG Tag Antibodies
GST Tag Antibodies
HA Tag Antibodies
His Tag Antibodies
Myc Tag Antibodies
TAP Tag Antibodies
V5 Tag Antibodies
【GAPDH】或G3PDH是甘油醛-3-磷酸脱氢酶（ glyceraldehyde-3-phosphate dehydrogenase ）的英文缩写。GAPDH是参与糖酵解的一种关键酶，由4个30-40kDa的亚基组成，分子量146kDa，检测条带大约在36kDa。GAPDH基因几乎在所有组织中都高水平表达，且GAPDH 作为管家基因在同种细胞或者组织中的蛋白质表达量一般是恒定的。
【Western Blot】蛋白质印迹法（免疫印迹试验）。它是分子生物学、生物化学和免疫遗传学中常用的一种实验方法。其基本原理是通过特异性抗体对凝胶电泳处理过的细胞或生物组织样品进行着色。通过分析着色的位置和着色深度获得特定蛋白质在所分析的细胞或组织中表达情况的信息。
β-Actin作为内参是得到了公认的，这是针对大多数组织和细胞来说的，它广泛分布于细胞浆内，表达量非常丰富。Beta-actin由375个氨基酸组成，分子量大小为42-43kDa左右。
β-actin的蛋白水平通常不会发生改变，因此被广泛用于Western时上样量是否一致的参照，也常被用于免疫染色观察细胞的微丝结构。在用作Western的参照时，Actin抗体和Tubulin抗体的主要不同之处在于两者所识别蛋白的分子量不同，这样可以选择合适的参照在同一块胶同一张膜上实现同时检测目标蛋白和参照蛋白。向左转|向右转
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