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.
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我用conA和LPS分别刺激脾细胞，当然细胞均有明显的增殖，可是如果把这两种有丝分裂原同时用来刺激细胞时，细胞的增殖明显受到了抑制，比起单独采用ConA或者LPS都低很多。conA和LPS分别刺激T细胞和B细胞的增殖，两者同时加入时，作用应该更强，为什么反而大幅减弱呢？
此外，我研究的这种成分单独刺激皮细胞时，细胞也有一定程度的增殖，但是如果这种物质与conA联合使用时，比起单独用conA时，细胞的增殖程度明显降低了一些，这又是什么原因呢？难道可以解释为我研究的这种物质有类似于LPS的作用——刺激B细胞增殖？
如果真的是这样来解释，哪位大侠能把第一个现象（conA和LPS共同作用产生拮抗）的机理帮我解释一下呢？
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。
抑制剂刺激细胞后，需要用PBS清洗后再做后续实验吗
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！！
