All calcium imaging experiments were performed on female flies ~1–5 days post-eclosion, and at room temperature. All physiology occurred within the animal’s ZT0 and ZT8. Animals of the proper genotype were collected and briefly anesthetized on ice. Once anesthetized, an animal was affixed to a custom-built holder with UV curable glue (BONDIC, M/N: SK8024). Our custom-built holder consists of a sheet of aluminum foil with a ~1 × 1 mm square (the imaging window) affixed to a 3D-printed design derived from similar designs described previously150 (link). Once mounted, a small window exposing the dorsal side of the brain was created, and covered with twice-filtered recording saline (in mM: 2 CaCl2, 5 KCl, 5 HEPES, 8.2 MgCl2, 108 NaCl, 4 NaHCO3, 1 NaH2PO4, 10 sucrose, and 5 trehalose; adjusted pH: ~7.4)29 (link). After establishing the imaging window, the air sacs, fat bodies, and trachea covering the dorsal side of the brain - as well as Muscle 16 - were removed with fine forceps. With the exception of minimal epochs during the synthetic MIP bath application experiments (see below), the brain was continuously perfused with oxygenated (95%O2/5%CO2) recording saline using a Cole-Parmer Masterflex C/L (M/N: 77120-62) at a rate of ~2 mL min−1.
Masterflex c l
Manufactured by Cole-Parmer
Sourced in United States
The Masterflex C/L is a peristaltic pump designed for laboratory applications. It features a variable-speed drive and a compact design. The pump utilizes interchangeable pump heads to accommodate a range of tubing sizes.
Automatically generated - may contain errors
Lab products found in correlation
Process 11, by Thermo Fisher Scientific (2 mentions)
Agarose type 7, by Merck Group (1 mentions)
Vt1000, by Leica (1 mentions)
Halothane, by Merck Group (1 mentions)
Ingeo biopolymer 7001d, by NatureWorks (1 mentions)
Ecoflex f blend c1200, by BASF (1 mentions)
Tcs sp8 inverted microscope, by Leica (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
9 protocols using masterflex c l
1
Calcium Imaging in Drosophila Brains
2
Perfusion of Brain Slices in ACSF
3
Rat Retina Slice Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rats were anesthetized by halothane (Sigma, St. Louis, MO, USA) inhalation and sacrificed by decapitation. Eyes were quickly removed and submerged in extracellular solution containing (in mM): 119 NaCl, 23 NaHCO3, 1.25 NaH2PO4, 2.5 KCl, 2.5 CaCl2, 1.5 MgSO4, 20 glucose and 2 sodium pyruvate. The solution was continuously aerated with 95% O2 and 5% CO2, reaching a pH of 7.4. Eyes were enucleated and the retina was carefully separated from the sclera. A small piece of retina was embedded in type VII agarose (Sigma) dissolved in a solution containing (in mM): 119 NaCl, 24 HEPES, 1.25 NaH2PO4, 2.5 KCl, 2.5 CaCl2, 1.5 MgSO4, pH 7.4, and was glued to the vibratome stage. Retinal slices of 200 µm thickness were made with a vibrating blade microtome (VT1000S, Leica Microsystems, Nussloch, Germany) and maintained in a chamber with oxygenated extracellular solution at room temperature (20°C) and photopic background illumination (100 lux). Retinal slices were then transferred to the recording chamber, held by a U-shaped platinum wire, and superfused with oxygenated extracellular solution at a rate of 1 ml/min, controlled by a peristaltic pump (Masterflex C/L, Cole-Parmer Instruments, Illinois, USA).
4
Biodegradable Fiber Elaboration from PLA, PBAT, and Cactus Stem
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The methodology for fiber elaboration was proposed by Black-Solis et al. [12 (link)] and Correa-Pacheco et al. [13 (link)]. The fibers were extruded from a mixture of two biodegradable polymers: PLA (IngeoTM Biopolymer 7001D, NatureWorks, LLC, Blair, NE, USA) and PBAT (Ecoflex® F Blend C1200, BASF, Mexico City, Mexico) in a 60/40 ratio (PLA/PBAT), and cactus stem flour at 3% using canola oil (Valley Foods®, Michoacán, Mexico) at 4% as a plasticizer. For the extrusion, a twin-screw extruder (Process 11, Thermo Scientific™, Waltham, MA, USA) was used with a temperature profile of 160/160/170/180/190/190/160 °C. The fibers were then cooled in water. The 60/40 pellets were dried at 60 °C for 24 h in a conventional oven prior to extrusion. A peristaltic pump (MasterFlex C/L, Cole-Parmer, Vernon Hills, IL, USA) was used for the addition of the cactus stem flour to the second port of the extruder.
5
Electrochemical Hydrogen Peroxide Generation
6
Microfluidic Bioreactor Platform Design
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The fluidic circuit consists of a 125 ml flask reservoir, a 12 V 50 rpm peristaltic pump (INTLLAB), a micro-peristaltic pump (marked as “precision pump” in Fig. 2 , Masterflex C/L, Cole-Parmer), and a microfluidic channel (details below). These components are interconnected using soft ¼” OD, 3/16” ID Tygon tubing (Cole-Parmer) with 1/16” OD PEEK tubing (IDEX) used to connect to microfluidic channel. Proper PEEK fittings (IDEX) connect the PEEK tubing to polypropylene barbed fittings (Cole-Parmer) connected to the Tygon tubing. Two flow loops are constructed as shown in Fig. 2 ; one “culturing loop” which bypasses the microchannel, and the observation loop which does flow through the channel. A polycarbonate stopcock is connected to the “culturing loop” to provide access to the fluidic circuit for either inoculation or sampling. A chemostat can also be integrated into the circuit to control microbe concentrations in the eChip platform in conjunction with the reservoir.
7
Fluorescence Microscopy Imaging Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed with 3.7% formaldehyde for 10 min at room temp, permeabilized and blocked with 0.5× TBS startblock (37542; Thermo Fisher Scientific) + 0.3% Triton-X100 for 0.5–1 h room temp. Cells were then stained 4°C overnight in antibodies (See Table S6 ) diluted in 0.25× startblock + 0.1% Triton-X100. After 10 min washing in PBS, cells were subjected to 1–2 h staining in secondary antibody diluted in 0.25× startblock + 0.1% Triton-X100 before mounting in Fluoromount-G (Southern Biotech) and imaging. Widefield fluorescence microscopy was performed with a Nikon 80i equipped with 10× (NA 0.45), 20× (NA 0.75), and 60× (NA 1.40) objectives and a CCD camera (Retiga 200R; QImaging). Where indicated, spinning-disk confocal microscopy was performed with a Leica TCS SP8 inverted microscope using a 63× (NA 1.4) oil immersion objective.
Live cell imaging experiments were performed using chambers (1µm-Slide_I_0.4; Ibidi) perfused with a peristaltic pump (Masterflex C/L; Cole Parmer) and pressure dampener in an environmental chamber mounted upon a spinning disc confocal microscope (Leica sp8). For roGFP experiments, 3 × 3 or 5 × 5 fields were imaged within the middle of chambers with a 60× oil immersion objective. For mito-Keima experiments, confocal stacks were acquired using 20× air and 63× oil objectives with Ex/Em of both 405/590 and 552/590.
Live cell imaging experiments were performed using chambers (1µm-Slide_I_0.4; Ibidi) perfused with a peristaltic pump (Masterflex C/L; Cole Parmer) and pressure dampener in an environmental chamber mounted upon a spinning disc confocal microscope (Leica sp8). For roGFP experiments, 3 × 3 or 5 × 5 fields were imaged within the middle of chambers with a 60× oil immersion objective. For mito-Keima experiments, confocal stacks were acquired using 20× air and 63× oil objectives with Ex/Em of both 405/590 and 552/590.
8
Fabrication of PLA/PBAT Biobased Fibers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fibers were prepared using a twin-screw extruder (Process 11, Thermo Scientific™, Waltham, MA, USA). The temperature profile was 160/170/170/180/180/190/190/160 °C from the feeding zone to the die. The outlet nozzle was 2.5 mm. The fibers were cooled in water. The composition of the polymer blend was PLA/PBAT 60/40 based on previous studies [13 (link)]. To add the compatibilizer and the plasticizers to the polymer blend in the extruder, an emulsion based on Tween 80 (0.5%), PPF (3%), CO (4%), and AA (7%) was prepared considering two temperatures, 27 ± 2 °C and 60 ± 2 °C, which was added to the extruder using a peristaltic pump (MasterFlex C/L, Cole-Parmer, Vernon Hills, IL, USA). The feeding speed was 0.15 mL/min. In total, six fibers were extruded: neat PLA and PBAT, PLA/PBAT, PLA/PBAT/CO, PLA/PBAT/PPF/CO/AA without emulsion heating or temperature (PLA/PBAT/PPF/CO/AA) and without emulsion heating (PLA/PBAT/PPF/CO/AA_T). The fiber composition is explained in Table 1 .
9
Macrophage Adhesion Dynamics in Flow
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!