110-day-old male flies were imaged before and after bolus injection of 10 mM EGTA or 100 µM valinomycin (Sigma-Aldrich, St. Louis, MO) containing artificial hemolymph-like solution (AHLS): 113 Na+, 5 K+, 8.2 Mg2+, 2 Ca2+, 133 Cl−, 5 HEPES, 4 HCO3−, 1 H2PO4,−, 10 Sucrose, 5 Trehalose, pH 7.5 (in mM). Borosilicate glass pipettes (1 mm OD, 0.75 mm ID, A-M Systems, Sequim, WA) were pulled using a P-1000 puller (Sutter Instruments). Pipette tip diameters of 50–75 µm were created by crushing the taper with forceps and visually confirming their diameter using a microforge (Narishige MF-830). Bolus injections into the abdomen of flies under ice anesthesia were approximately 1000 pL in volume and were made using a Femtojet (Eppendorf, Hamburg, Germany), with the pipette positioned using a manual micromanipulator (World Precision Instruments, Sarasota, FL).
Manual micromanipulator
Manufactured by World Precision Instruments
The manual micromanipulator is a precision positioning device used to delicately control the movement of microscopic objects or tools. It offers fine, incremental adjustments along multiple axes to facilitate accurate positioning and manipulation in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Valinomycin, by Merck Group (1 mentions)
P 1000 puller, by Sutter Instruments (1 mentions)
Mf 830, by Narishige (1 mentions)
Femtojet, by Eppendorf (1 mentions)
Ms 222, by Merck Group (1 mentions)
Picospritzer, by Parker Hannifin (1 mentions)
Microsyringe pump controller, by World Precision Instruments (1 mentions)
Fk 506 rapamycin, by LC Laboratories (1 mentions)
4 protocols using manual micromanipulator
1
Hemolymph ion manipulation and imaging
2
Targeted Eye Electroporation in Xenopus
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Targeted eye electroporation can be performed as previously described (54 (link), 55 (link)). To anesthetize embryos during electroporation, tricaine methanesulfonate (MS-222) (0.4 mg/ml; Sigma-Aldrich) in 1× Modified Barth’s Saline (MBS) [LM00715 (male), LM00535 (female), Nasco] (pH 7.5) is used. An anesthetized embryo is positioned along the longitudinal channel of a “†”-shaped electroporation Sylgard chamber, with its head positioned at the cross of the longitudinal and transverse channels. A pair of flat-ended platinum electrodes (Sigma-Aldrich) is held in place by a manual micromanipulator (World Precision Instruments) at the ends of the transverse channel. A glass capillary with a fine tip containing plasmid solution is inserted into the eye primordium of stage 26–30 embryos to inject 8 × 5- to 8-nl doses of pEGFPC1-hVAP-A (1 μg/μl; Addgene #104447) or pEGFPC1-hVAP-A KD/MD (Addgene #104449) plasmid driven by an air-pressured injector, such as a Picospritzer (Parker Hannifin). Immediately following the plasmid injection, eight electric pulses of 50-ms duration at 1000-ms intervals are delivered at 18 V by a square wave generator, such as the TSS20 Ovodyne electroporator (Intracel). The embryos are recovered and raised in 0.1× MBS until they reach stage 32–35 as required for retinal cultures.
3
Quantifying Hydrogen Peroxide Release in LPS-Stimulated Cells
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Each 24-well plate was removed from the incubator and partially immersed in a 37 °C water bath (VWR). The Pt MEA was secured to a manual micromanipulator (World Precision Instruments), which was positioned such that the probe was inserted into the culture medium and secured 5 mm above the bottom surface of the plate as shown in Figure 4 . Using the mPD-coated platinum microelectrode array (Pt MEA) with the FAST-16mkIII potentiostat in a two-electrode configuration, we performed chronoamperometry at an applied potential of +0.7 V with reference to an Ag/AgCl electrode. Current measurements were taken at a frequency of 10 Hz. The FAST system software (Quanteon) was used in all chronoamperometry experiments.
As proof of concept for detection of H2O2 release, RAW 264.7 cells were incubated at 37 °C, 5% CO2 until they reached ~50% confluency, then doped with 200 ng or 500 ng LPS. After further incubation for 24 h under the same conditions, the cells were probed.
As proof of concept for detection of H2O2 release, RAW 264.7 cells were incubated at 37 °C, 5% CO2 until they reached ~50% confluency, then doped with 200 ng or 500 ng LPS. After further incubation for 24 h under the same conditions, the cells were probed.
4
Intraspinal Transplantation of Human Cells
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