Screening and imaging of Tg(cxcl18b:eGFP) or Tg(mpx:GFP) zebrafish larvae were performed with a Zeiss Axio Zoom V16 stereo microscope with a Hamamatsu ORCA flash 4.0 camera attached. Time-lapse confocal images were acquired with a Nikon A1R confocal microscope. Tg(cxcl18b:eGFP) zebrafish larvae were imaged 1 h after injection into the hindbrain ventricle with 2,000 CFU of S. pneumoniae PLY+, S. pneumoniae PLY- or PBS. Images were obtained at 1 h intervals for 20 h. Tg(mpx:GFP) casper zebrafish larvae were imaged 1 hpi in the hindbrain ventricle with 2,000 CFU of mCherry labeled S. pneumoniae D39V and images were obtained at 30 min intervals for 9 h. After injection, the anesthetized larvae were embedded in 1.5% low-melting point agarose dissolved in egg water dissolved in egg water (60 μg/mL sea salts (Sigma-Aldrich, S9883) in MiliQ) with 0.02% (w/v) Tricaine (Sigma-Aldrich, Cat# A5040) and GSK′872 (RIPK3 inhibitor) (MedChemExpress; Cat# HY-101872) in 1% DMSO was added to a final concentration of 100 μM or 1% DMSO alone (vehicle control) in an open uncoated 8-well chamber slide (Ibidi, Cat# 80826) and kept at 28°C in a temperature-controlled chamber (Okolab). NIS Elements and ImageJ were used for image analysis and neutrophil quantification.
Temperature controlled chamber
Manufactured by Okolab
The Temperature-controlled chamber is a device designed to maintain a specific temperature environment. It provides precise temperature regulation to create a controlled environment for various applications.
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2 protocols using temperature controlled chamber
1
Zebrafish Infection Imaging Protocol
2
TIRF Microscopy for Agonist-Driven GRK2 Recruitment
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To monitor agonist-driven plasma membrane recruitment of GRK2, we used a TIRF microscopy–based assay described previously (27 (link)). Cells were washed and live imaged in Hepes-buffered saline imaging solution with 135 mM NaCl, 5 mM KCl, 0.4 mM MgCl2,1.8 mM CaCl2, 20 mM Hepes, and 5 mM d -glucose adjusted to pH 7.4. Time lapse image series were acquired at 37°C with 488-nm laser excitation using a 100× 1.49 oil CFI Apochromat TIRF objective on a Nikon TIRF microscope operated with NIS Element AR 5.21.03 and equipped with a temperature-controlled chamber (Okolab), perfect focus system, and an Orca Fusion BT sCMOS camera at 5-s intervals. Drugs were added by bath application at concentrations indicated in the figure legends. Protein relocalization was calculated as R(t)/R0 with R(t) being the fluorescence signal at each time point (t) and R0 being the mean fluorescence signal of the 10 time points before agonist addition.
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