Microforging, micropipette calibration and microinjection — in a single device!
- Microprocessor-controlled microforge
- Digital signal processor technology precisely controls the polish heating time
- Unique digital pneumatic pressure feature polishes the tip without changing the size
Options
| Order code | Options |
| DMF1000-1 | 110 V,Microscope |
| DMF1000-M1 | 110 V, without Microscope |
| DMF1000-2 | 220 V,Microscope |
| DMF1000-M2 | 220 V, without Microscope |
Click here to view the current Data Sheet.
Click to view the pullers, bevelers, microforge application guide to compare all the units.
Benefits
- Digital Signal Processor (DSP) technology
- Complete system package available
- Kohler illuminator and Abbe condenser for less glare and sharper images.
- Pneumatic pressure polishing that allows the preparation of blunt tips without change of tip ID
- Heating filament is attached to the microscope objective so they move together
- Pipette holder sits on the microscope stage to simplify the locating and polishing of the pipette
Applications
- Polishing patch pipettes
- Microforging holding pipettes
- Microforging beveled injection pipettes
- Pipette tip calibration and microinjection
The DMF1000 is a microprocessor-controlled microforge offering unmatched performance. Designed for fabrication of both small patch clamp glass pipettes and larger injection pipettes, the DMF1000 should find many uses in the laboratory. The DMF1000 is based on a design similar to that first used in WPI’s extremely popular microforge model, the MF200. The extensive improvements incorporated into the DMF1000 greatly increase its versatility and performance, making it one of the most powerful microforges on the market.
Digital Signal Processor (DSP) Technology
The DMF1000 is powered by the latest digital signal processor (DSP) technology. A digital timer is used to precisely control the polish heating time. Ten memories can be used to store settings of the heating power and heating duration. All of the settings are controlled and displayed digitally for better accuracy and reproducibility. Two different operating modes are provided: Manual and Auto. In the Manual mode, the DSP will memorize the duration of the time that is used to achieve a desired polishing. In Auto mode, the heat will be applied for the duration of the timer setting.
Complete System Available
The DMF1000 system includes a specially configured WPI model W30S-LED research grade compound microscope (optional) equipped with a high quality metallurgic 40x long-working distance objective and a pair of 10x eyepieces. The long working distance objective reduces the danger of damage to the objective lens during the heating process.
Kohler Illuminator and Abbe Condenser
Other benefits of the DMF1000 design include the use of a Kohler illuminator and Abbe condenser, which provide the reduced glare and sharper image contrast necessary when polishing pipettes as small as half a micron (0.5 µm) in diameter.
Pressure Polishing
The DMF1000 incorporates a unique digital pneumatic pressure feature that enables pressurized air to be delivered through the pipette during fire polishing. In the fabrication of patch pipettes, the pressurized air can be used to blunt the taper at the pipette tip without changing the size of the tip opening. This reduces electrical resistance of the tip, leading to lower noise during patch-clamp recordings (Goodman & Lockery, 2000).
The Heating Filament
With a conventional microforge often the most difficult and time-consuming part of using a high magnification objective is being able to move both the heating filament and the pipette into the same viewing area. Finding and moving both the heating filament and the pipette without collision can be a challenge. However, this difficulty is eliminated with the DMF1000 because the heating filament is directly attached to the microscope’s objective. Hence it can be easily adjusted to any position within the viewing area.
The low heat capacity and low thermal coefficient of linear expansion of the filaments are key design features. The low heat capacity of the filament allows it to reach fire-polishing temperatures without excessive heat. This permits you to bring the pipette tip close to the filament during polishing without fear of collapsing the pipette tip. Low heat capacity eliminates the need for an auxiliary air-cooling system. The low coefficient of expansion characteristic of the filament ensures minimal displacement of the filament during heating. This feature eliminates much of the guesswork out of tip placement in relation to the filament.
Two different heating filaments are provided to accommodate various applications. The H5 filament is large gauge and can be reformed into a “U” for fabrication of pipettes, air forming of patch pipettes and other applications. The H4 is a smaller gauge filament and is ideal for polishing patch clamp pipettes.
Pipette Holder Sits on the Microscope Stage
The pipette rests on a specially designed holder that sits on top of the microscope stage. The position of the pipette, relative to the heating filament, is controlled by the (X, Y, Z) adjustment of the stage. This unique design makes locating and polishing the pipette extremely easy. The stage of the microscope has a high quality rail that gives precise, smooth and stable control of the pipette"s movement. This configuration also eliminates the need and expense of an additional micromanipulator to control pipette movement.
Typical applications of the DMF1000
Polishing the Patch Pipettes
Proper fire polishing of patch pipettes is the single most important factor for forming a stable giga-seal in patch clamp recording. This is even more important than the type of glass capillary used. Difficulties often arise in forming giga-seals because the polishing of patch pipettes using a conventional low magnification microforge is inadequate. However, the DMF1000 uses a 40X long-working distance objective. Pipette polishing is much more accurately controlled. Both whole cell patch pipettes and single channel patch pipettes can be conveniently polished to the highest quality and reproducibility achievable with any microforge.
Microforging Holding Pipettes
A holding pipette with a large blunt tip and a small opening is used to hold a floating cell in place prior to microinjection by applying suction to the rear of the pipette. The procedure for making holding pipettes involves three steps: squaring off, large bore flame polishing and tip reducing. These steps are accomplished with a larger heating filament.
Microforging Beveled Injection Pipettes
Occasionally, a beveled large bore pipette is not sharp enough to penetrate a cell without damaging the area around the pipette. With the DMF1000 and the large heating filament, a sharp point can be formed on the beveled tip to assist the penetration of the cell. This process is referred to as contact stretching.
Pipette Tip Calibration & Microinjection
The integrated digital pneumatic pressure system can be used to calibrate the precise diameter (I.D.) of a micropipette tip, based on a technique described previously (Hagag & Randolph 1990, Bowman & Ruknudin 1999). The pressure system can also be used separately as a simple but highly accurate controller for microinjection applications.
DMF1000 Instruction Manual
| AC POWER MODULE | 100-240 VAC 50/60 Hz |
| TIMER RANGE (for heater & timer) | 0.01 to 360 sec |
| NUMBER OF MEMORYS | 10 |
| PRESSURE ADJUSTING RANGE | 0.5 - 60 PSI (3.5 -414 kPa) |
| PRESSURE RESOLUTION | 0.1 PSI (0.7 kPa) |
| FILAMENTS: H4 | Small filament for working with 40x long working distance objective. |
| FILAMENTS: H5 | Large filament for working with 10x objective. Filament adjustment assembly provided for both objectives. |
| HEATER AND TIMER CONTROL | Auto or Manual via Pushbutton, TTL, or Optional foot switch |
| DIMENSIONS: Control Unit | 4 x 7 x 17 in. (10.2 x 17.8 x 4.8 cm) |
| SHIPPING WEIGHT | 4 lb. (1.8 kg) |
| MICROSCOPE | W30S |
| MICROSCOPE: SHIPPING WEIGHT | 16 lb. (7.3 kg) |
Wu, Z.-Z., Chen, S.-R., & Pan, H.-L. (n.d.). Differential Sensitivity of N-and P/Q-Type Ca2+ Channel Currents to a ? Opioid in Isolectin B 4 -Positive and -Negative Dorsal Root Ganglion Neurons. http://doi.org/10.1124/jpet.104.073429
Replacement Filament Cable75040For pricing, Customers outside of the US and Canada, please contact your distributor.
Replacement Micropipette Slide75050For pricing, Customers outside of the US and Canada, please contact your distributor.
40X Long Working Distance Objective, 3mm 0.25 NA800292For pricing, Customers outside of the US and Canada, please contact your distributor.
Eyepiece with 100/10 reticle for W30S microscope503513For pricing, Customers outside of the US and Canada, please contact your distributor.
Replacement Heating Filament (large gauge) for DMF1000 MicroforgDMF1000-H5For pricing, Customers outside of the US and Canada, please contact your distributor.
Optional Foot Switch for Micro2T, Micro-ePore™, DMF100013142For pricing, Customers outside of the US and Canada, please contact your distributor.
ebiomall.com
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1. 姜黄素是一个典型的HAT抑制剂。
2. 针对P300: 在大约10年前，Cole和他的同事设计出了一种p300/CBP抑制剂，发表在nature杂志上。
希望能帮到你，望采纳！
支原体培养则是取样后在培养基上培养，看有多少支原体菌落会长出，是比较直观和可信的结果。
总体来讲，这两种检查手段可信度都较高，结合一起，不仅可以可靠的知道有无解脲支原体感染，还能知道感染是否严重。
该试剂盒是一种时间分辨的荧光共振能量转移免疫分析，该反应是一个竞争免疫反应，即铕标的cAMP示踪复合物与体系中的cAMP竞争结合标有Alexa Fluor 647染料的cAMP抗体。铕标cAMP示踪复合物是通过Biotin标记的cAMP与铕标的抗生物素蛋白链菌素（streptavidin）与抗体的复合物紧密结合产生的。
当抗体结合到示踪剂上时，340nm的激发光激发铕标分子，导致能量转移到Alexa Fluor 647染料上，结果产生665nm的发射光。荧光的强度与样品中的cAMP含量成反比。
本试剂盒用于检测在GPCR激动剂刺激下活细胞或者细胞膜制备品产生的cAMP。对于偶联Gαs的受体，激动剂刺激导致665nm的荧光强度降低，而拮抗剂则可以逆转这一效应；对于偶联Gαi的受体，在激动剂刺激的同时用forskolin刺激cAMP产生，那么激动剂则抑制forskolin诱导的cAMP的生成，因此对照只给forskolin的细胞组可以通过665nm荧光强度的增加反应激动剂的效应。
该试剂盒的灵敏度很高，室温下反应在20h内是稳定的。本试剂盒适用于在384孔板中进行24μl的微量分析。
2.保存条件
避光2~4℃保存，过期时间见外包装。
3.盒内试剂
cAMP标准品：1管，1ml。（50μM）
生物素标记的cAMP（b-cAMP）：1管，25μl。
铕标的抗生物素蛋白链菌素：1管，25μl。
荧光标记的cAMP抗体：1管，40μl。
检测缓冲液：1瓶，25ml。
4.需要自配的其他溶液
l Hank’s balanced salt solution (HBSS): NaCl 8.0g、CaCl2 0.14g、KCl 0.4g、 KH2PO4 0.06g、Na2HPO4?7H2O0.09g、MgCl2.6H2O0.10 g、MgSO4.7H2O0.10 g、NaHCO30.35g、葡萄糖1.0g，加H2O至 1000ml (用7.5%NaHCO调节PH值=7.4)
l Versene消化液（1L）：EDTA 0.372 g，NaCl 8.0g，KCl 0.20 g，KH2PO40.20g，Na2HPO4 1.15 g，D-glucouse 0.2 g，pH 7.4
l HEPES缓冲液(1mol/L)：取2.383gHEPES溶于10ml去离子水中。
l 7.5%BSA溶液：取0.75gBSA溶于10ml去离子水中
l 0.5M IBMX溶液：11.11mg IBMX溶于100μl DMSO中，-20℃冻存。
l 刺激缓冲液（SB）：14 ml HBSS(1×)+75μlHEPES(1mol/L)+200μlBSA (7.5%)。(注：在测定细胞cAMP时，反应缓冲液中要加入IBMX 0.5mmol/L)
l 吗啡贮存液(10mM)：盐酸吗啡37.585mg溶于10ml生理盐水中，0.22μm滤膜过滤除菌，4℃保存备用。
l 纳络酮母液(100mM)：纳络酮4mg溶于100μl 生理盐水中，用时工作液按照1:500稀释，溶剂为含有IBMX的反应缓冲液。
我做的是细胞因子的刺激和抑制某条通路后观察是否有影响，分组为空白组，空白+抑制剂，刺激组，刺激+抑制剂，最开始用的单因素方差分析，LSD-T和SNK-Q检验，但是同学说我这里面有两个处理因素，所以不能单因素方差分析，应该直接空白和空白+抑制，空白和刺激，刺激和刺激+抑制剂进行独立样本T检验，现在脑子是混乱的，拜托园子里的大神们帮我看看，感激不尽！！
成纤维细胞生长抑制试剂盒—FibrOut™ 是由多种生化复合物组成的混合物、抗体或特殊的试剂。它能够抑制成纤维细胞过度增长或污染从而有效地增加靶细胞的产量。每种成纤维细胞生长抑制试剂盒都可定制到特定组织和特定细胞，经验证可与对应的PrimaCellTM系统高效协同工作，可为您带来每一类原代细胞培养的最佳结果。每种成纤维细胞生长抑制试剂盒都配备了最合适的缓冲液。
FibrOut™ 是一种包含几种生化复合物和试剂的完整系统，它在原代细胞培养中能抑制成纤维细胞增殖，从而促进靶细胞生长。每种成纤维细胞生长抑制试剂盒都可定制到特定组织和特定细胞，经验证可与对应原代细胞培养试剂盒-PrimaCellTM高效协同工作，可为您带来每一类原代细胞培养的最好结果。每种成纤维细胞生长抑制试剂盒都配备了最合适的缓冲液。成分包括 (1)胰蛋白酶；(2)胶原酶；(3)D-缬氨酸；(4)顺式羟基脯氨酸；(5)硫柳汞；(6)苯巴比妥；(7)系列血清替代品；(8)抗中胚层带抗体。
参考文献：
1. Cancer Res. 2010 May 1;70(9):3537-46，Epub 2010 Apr 20. Requirement of the NF-kappaB subunit p65/RelA for K-Ras-induced lung tumorigenesis. Bassères DS, Ebbs A, Levantini E, Baldwin AS.
2. Nat Commun. 2013;4:1795. Recruitment of mesenchymal stem cells into prostate tumours promotes metastasis. Jung Y, Kim JK, Shiozawa Y, Wang J, Mishra A, Joseph J, Berry JE, McGee S, Lee E, Sun H, Wang J, Jin T, Zhang H, Dai J, Krebsbach PH, Keller ET, Pienta KJ, Taichman RS.
3. Mol Biol Cell. 2012 Jul;23(14):2755-69. Calcium-calmodulin kinase I cooperatively regulates nucleocytoplasmic shuttling of CCTα by accessing a nuclear export signal. Agassandian M, Chen BB, Pulijala R, Kaercher L, Glasser JR, Mallampalli RK.展开
、
Cell-Based ELISA
的优点：
Cell-Based ELISA
（基于细胞的
ELISA
）是一种全新的
ELISA
技术，有两个最突出的优点：
1.1
不需要抽提蛋白、包被微孔板：细胞直接在微孔板里培养，待检测的时候，将细胞固定在微孔板上并
进行通透处理即可。这样就避免了抽提蛋白时，由于客观和主观上引起样品的损失而导致实验结果在一定
程度上偏离了实际情况。
同时不用包被微孔板，
简化了实验流程，
有助于提高效率。
科研人员在一个
96
孔
酶联板上，便能检测目标细胞蛋白经刺激或抑制作用后的表现。由于省去抽提蛋白和裂解细胞的步骤，样
本的损失也能降到最低，比起其他普通的
ELISA
测定方法，这项全新的
ELISA
技术能更快速、更方便
地一次检测大量的细胞内蛋白。
1.2
可同时检测两种不同蛋白：
封闭后加入两种抗不同蛋白且来源于不同宿主的一抗，
然后再加入不同的
二抗，加入两种荧光底物，检测两个波长。同时检测两种蛋白的好处是显而易见的，可以减少工作量，此
外还可以满足一些特殊的实验需要，例如，需要测定某个蛋白的磷酸化比例，就需要测定磷酸化蛋白的数
量和总蛋白的数量，这两个测定在同一次实验进行，有助于消除实验误差，得到更为精确的实验结果。
2
、
Cell-Based ELISA
的两种技术：
某些公司发展了双通道
Cell-Based ELISA
技术；
双通道与单通道
Cell-based ELISA
比较：
双通道
cell-based ELISA
，顾名思义，即一次可以同时检测两种目的蛋白，
R&D Systems
提供的
Cell-based ELISA
就是双通道
cell-based ELISA
，原理略：（需要荧光检测方式和相应仪器）；
单通道
cell-based ELISA
，即一次只能检测一种目的蛋白，他和普通
ELISA
的主要区别在于样品处理过
程
3
、
cell-based ELISA
的应用：
该产品，最多发展起来的是用于检测磷酸化和非磷酸化蛋白的相对含量；
4
、有
cell-based ELISA
产品的公司：目前，多家
elisa
产品提供
商均
提供
cell
based
elisa
试剂盒，
如
Rnd
systems
，
raybiotech
，
ebioscience
，
millipore
等公司；
5
、如何自己进行
cell based elisa
实验？
事实上，根据单通道的
cell based elisa
原理，可以自己建立
cell based elisa
实验系统；
细胞加入培养板中；
加入刺激物或者抑制剂进行培养；
对细胞进行固定或者封闭；
加入第一抗体；
加入
HRP
结合的二抗（二抗的选择，同
western blot
）；
加入底物进行显色；
2.微孔板：选择实验所需的板条数。剩余不用的部分随同干燥剂密封放回原袋。 1.包被有抗抑制素bB亚单位抗体的微孔板（Anti-InhibinB-CoatedMicrotitrationStrips）：96孔，2-8ºC下密封保存。
2.标准品A/样本稀释液（InhibinBStandardA/SampleDiluent）：2ml×1瓶，胎牛血清，含0pg/mL二聚体抑制素B。使用前2-8ºC下保存。打开后2-8ºC下可保存2周。于-20ºC可长期保存。
3.标准品B-G（InhibinBStandardsB-G）：1ml×6瓶，胎牛血清，含二聚体抑制素B的浓度为10,30,100,250,500,1000pg/mL。使用前2-8ºC下保存。打开后2-8ºC下可保存2周。于-20ºC可长期保存。
4.质控（InhibinBControls）：1ml×2瓶，浓度I、II，胎牛血清，含低浓度和高浓度的抑制素A二聚体。使用前2-8ºC下保存。打开后2-8ºC下可保存2周。于-20ºC可长期保存。
5.样本稀释液A（InhibinBSampleBufferA）：10ml×1瓶，2-8ºC下保存。
6.样本稀释液B（InhibinBSampleBufferB）：10ml×1瓶，2-8ºC下保存。
7.抗体-生物素结合物（InhibinBAntibody-BiotinConjugate）（即用型）：10ml×1瓶，含生物素标记的抗抑制素a亚单位抗体。2-8ºC下保存。
8.酶结合物（浓缩）（Streptavidin-EnzymeConjugate）（即用型）：10ml×1瓶，含链霉和素-辣根过氧化物酶结合物。2-8ºC下保存。
9.TMB底物溶液（TMBChromogenSolution）：15ml×1瓶，2-8ºC下保存。
10.终止液（StoppingSolution）：15ml×1瓶，为0.2M的硫酸。2-8ºC下保存。
11.浓缩洗液（WashConcentrate）：100ml×1瓶，2-8ºC下保存。 操作前所有试剂都应平衡至室温(~25ºC)并充分混匀。标准品、质控及待测样本需同时做双份。
1.取出实验所需板条，并记录各孔位置。
2.在对应的孔中加入标准品、质控及待测样本各50ul。
3.每孔加入25ul样本稀释液A。
4.每孔加入25ul样本稀释液B。
5.封板，以300-400rpm速度震荡，室温下过夜（14-18小时）。
6.洗板3次，在吸水纸上拍干。
7.每孔加入50ul抗体-生物素结合物。
8.封板，以500-700rpm速度震荡，室温下孵育1.5小时。
9.洗板6次，在吸水纸上拍干。
10.每孔加入50ul链霉和素-辣根过氧化物酶结合物。
11.封板，以500-700rpm速度震荡，室温下孵育20分钟。
12.洗板6次，洗完最后一次以后，将洗液在孔内停留15分钟再除去。在吸水纸上拍干。
13.在每孔中加入100ulTMB底物溶液。
14.以500-700rpm速度震荡，室温下避光孵育15-30分钟。
15.每孔加入100ul终止液。
16.30分钟内在450nm处读数。
注：必须以零标准作空白。如果能做到双波长测定，可在600或620nm处读数，将600（620）nm吸收值从450nm处减去，这样可以减少光学误差。 使用血清样本，静脉穿刺术采集血液。样本于2-8ºC可保存24小时，-20ºC或以下可保存30天。避免反复冻融样本。不应采用溶血或脂血的样本。冰冻的样本在实验前应解冻，并充分混匀。向左转|向右转
