Cerebrospinal fluid was collected from the cisterna magna (see intra cisterna magna injection for animal preparation) under a dissection microscope using a glass capillary (Sutter Instrument, B100–50-10, pulled with a Sutter Instrument P-30 micropipette puller to a size of 0.5 mm in diameter). 10 μl of the obtained volume from two mice was pooled and used for the quantification of cytokines using Luminex magnetic beads following the Bio-Plex Pro Mouse Chemokine Panel 33-plex instruction (Bio-Rad). Data were acquired with a Bio-Plex 200 with HTF and analyzed with the Bio-Plex Manager software Version 6.1.
B100 50 10
Manufactured by Sutter Instruments
Sourced in United States
The B100–50-10 is a laboratory equipment product manufactured by Sutter Instruments. It serves as a power supply, providing a controlled electrical output for various scientific applications. The device specifications include an output range of 0-100V and 0-50mA, with a maximum power output of 10W.
Automatically generated - may contain errors
Lab products found in correlation
P 30 micropipette puller, by Sutter Instruments (4 mentions)
Bio plex pro mouse chemokine panel 33 plex, by Bio-Rad (2 mentions)
Bio plex 200, by Bio-Rad (2 mentions)
Bio plex pro mouse cytokine panel 23 plex instruction, by Bio-Rad (2 mentions)
Xponent software v4, by DiaSorin (2 mentions)
Flexmap 3d, by DiaSorin (2 mentions)
Bf100 50 10, by Sutter Instruments (1 mentions)
Af100 64 10, by Sutter Instruments (1 mentions)
Mf 900 microforge, by Narishige (1 mentions)
Ms 222, by Merck Group (1 mentions)
Pronase digestion, by Merck Group (1 mentions)
Low melting agarose, by Merck Group (1 mentions)
Stemi 508, by Zeiss (1 mentions)
P 1000, by Sutter Instruments (1 mentions)
7 protocols using b100 50 10
1
Cerebrospinal Fluid Cytokine Quantification
2
Cerebrospinal Fluid Collection and Cytokine Analysis
3
Optimized Microinjection Protocol
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Needles used in the microinjection procedure were freshly pulled using either borosilicate glass capillaries with filament (BF100-50-10, Sutter Instrument, USA) or aluminosilicate glass capillaries with filament (AF100-64-10, Sutter Instrument, USA) on a Sutter P-1000 micropipette puller (Sutter Instrument, USA) with the following settings: Heat = ramp-34, Pull = 50, Velocity = 70, Time = 200, Pressure = 460 for borosilicate glass and Heat = ramp, Pull = 60, Velocity = 60, Time = 250, Pressure = 500 for aluminosilicate glass. The tips of the needles were afterwards broken and sharpened using a MF-900 microforge (Narishige, Japan). Needles were loaded using either capillary motion or microloader tips (Eppendorf, Germany). Embryos were kept in position using glass holders pulled from borosilicate glass capillaries without a filament (B100-50-10, Sutter Instrument, USA) using P-1000 puller with the following settings: Heat = ramp + 18, Pull = 0, Velocity = 150, Time = 115, Pressure = 190. The holders were broken afterwards using a MF-900 microforge to create a tip of ~140 µm outer diameter and 50 µm inner diameter. Tips were heat-polished to create smooth edges and bent to a ~20° angle.
4
Zebrafish Blastomere Transplantation Assay
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Embryos from H2B-GFP, Ptf1a-dsRed double transgenic zebrafish were dechorionated by pronase digestion (0.6 mg/ml; Sigma) and placed in agarose molds (Adaptive Science Tools), and one to five blastomeres were transplanted into an unlabeled embryo at 3.5 hpf using a flame-pulled glass capillary (Sutter instruments, #b100-50-10) connected to a 2 ml syringe. The host embryos were allowed to recover at 32°C overnight in agarose-coated dishes in order to catch up developmentally. At 24 hpf, embryos were anaesthetized by 0.04% MS-222 (Sigma) and screened on an upright fluorescent microscope where isolated GFP-positive RPCs could be identified. Position and number of cells were logged before the fish were placed in individual wells at 28.5°C. At 72 hpf, embryos were fixed for 1 hr in 4% PFA, the eye dissected out, and mounted in 1% low melting agarose (Sigma) for imaging.
5
Microinjection protocol for nematode EVs
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A Warner Instruments microinjector (Pico-Liter Injector Model PLI-90A), PicoNozzle needle mount (World Precision Instruments, Inc; 5430-ALL), and Zeiss Stemi 508 were used for microinjections. Pressure to Pico-Liter Warner Injector was set at 40 psi, injection time at 0.20 sec, pressure meter at 1 psi, and pressure balance at −0.2 psi. Borosilicate glass capillaries (Sutter Instrument; B100-50-10) were pulled into needles on a needle puller (Sutter instruments P1000), using 563 for heat, 60 for pull, 80 for velocity, 90 for delay, 200 for pressure, 553 for ramp with delay mode and sufe heat settings on. Each needle was loaded with ~10 μl of an EV F1/dextran mixture (above, EVs from 1 animal per μl, approximately 4×1011 EVs total in each 100 μl EV preparation esimated from NTA experiments with identical animal numbers) or buffer/dextran control solution (no EVs). One animal at a time was poked with a fine blade (FST; 10055–12) between the tail branches on the dorsal side, then injected with ~1 μL (approximately 4×109 EVs) for RNAseq (larger animals, 6–7 mm) and ~0.25 μL (approximately 1 ×109 EVs) for anti-pH3-PS10 and EdU labeling (smaller animals, 4–5 mm). Animals were allowed to recover for at least one hour then worms without fluorescence were removed.
6
Cerebrospinal Fluid Cytokine Quantification
7
Cerebrospinal Fluid Collection and Cytokine Analysis
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Mice were anesthetized via i.p. administration of ketamine/xylazine and placed on a stereotactic frame. Cerebrospinal fluid was collected from the cisterna magna under a dissection microscope using a glass capillary (Sutter Instrument, B100–50–10, pulled with a Sutter Instrument P-30 micropipette puller to a size of 0.5 mm in diameter). CSF (12.5 μl ) was obtained from each mouse and analyte quantification performed using Luminex magnetic beads with the Bio-Plex Pro Mouse Cytokine Panel 23-plex instruction (Bio-Rad). Data were acquired with the Luminex Flexmap 3D and analyzed with xPONENT Software V4.2 (both from Luminex Corp., Austin, TX).
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