点评详情
发布于:2018-10-17 16:21:56  访问:0 次 回复:0 篇
版主管理 | 推荐 | 删除 | 删除并扣分
Or outside-out patch configuration with an EPC10 amplifier (HEKA) at {room
To reduce the C. (1998) Anthrax lethal element cleaves the N terminus resolution exchange time in the whole-cell configuration, currents were recorded from modest HEK293 cells, showing a membrane capacitance of 8 ?6 picofarads. Briefly, the mouse was sacrificed just after being deeply anesthetized with ketamine (one hundred mg/kg) and xylazine (10 mg/kg). The entire brain was removed and placed in iced and 95 O2, 5 CO2 oxygenated cutting medium such as the following (in mM): 230 sucrose, two.5 KCl, 10 MgSO4, 1.25 Na2HPO4, 26 NaHCO3, 0.5 CaCl2, ten D-glucose. The sagittal or coronal slices had been reduce using a VT1200S vibratome (Leica) and moved for the artificial cerebrospinal fluid (ACSF) saturated with 95 O2, five CO2. The ACSF contained the following (in mM): 124.Or outside-out patch configuration with an EPC10 amplifier (HEKA) at room temperature (22?five ). Recording microelectrodes with resistances of three? megohms have been pulled from thin-walled borosilicate glass with inner filament (Sutter Instrument) and filled using the intracellular answer containing 135 mM CsCl, 10 mM EGTA, four mM Mg-ATP, and ten mM HEPES (pH adjusted to 7.2 with CsOH, 290 ?95 mosM). The coverslips had been placed in a perfusion chamber and continuously perfused having a common extracellular remedy containing 150 mM NaCl, 5 mM KCl, 10 mM HEPES, 1 mM MgCl2, 2 mM CaCl2, and 10 mM glucose (pH adjusted to 7.35 with NaOH, 305?15 mosM). To ensure cell dialysis, measurements have been obtained at the least three? min right after the whole-cell configuration was established. Channel currents have been recorded in outside-out patch or lifted whole-cell configuration at a holding possible of 60 mV, and signals have been analog low passfiltered at 2.9 kHz (four-pole Bessel-type, 23 db) and sampled at 20 kHz. In experiments involving recording currents from heteromeric GluK2a/GluK5 receptors, we utilized the following established criteria (41, 42). First, the cells have been cotransfected with GluK2a and GluK5 cDNAs PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27463369 inside a 1:three ratio, which yielded a largely homogeneous population of GluK2a/GluK5 heteromeric KARs in the cell membrane surface (50); Second, glutamate pulses (1 mM, 100 ms) delivered to GluK2a/GluK5 cotransfected cells elicited current responses of smaller sized amplitude than that of GluK2a singly transfected cells. Third, the rectification index of heteromeric receptors is greater than 0.9 (Fig. 6B). Drug Application--Glutamate was applied to HEK293 cells using a speedy step perfusion system (Warner Instruments, SF-77B) via a -tube with a tip diameter about 200 m. Patches of outside-out or lifted whole cells have been positioned close to the solution interface, as well as the interface was moved by applying voltage across digital to analog converter output on EPC10 amplifier to SF-77B. The open-tip recordings with the liquid junction potentials revealed that the 10 ?0 exchange PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25936642 of resolution occurred within 300 s. For lifted whole-cell recording, HEK293 cells have been lifted in the coverslip and placed within 100 m of the tip of a tube (41, 43, 44). To lessen the answer exchange time in the whole-cell configuration, currents have been recorded from small HEK293 cells, displaying a membrane capacitance of eight ?six picofarads.
共0篇回复 每页10篇 页次:1/1
共0篇回复 每页10篇 页次:1/1
我要回复
回复内容
验 证 码
看不清?更换一张
匿名发表 
当前位置
脚注信息

美博梁sir2010-2017版权所有