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Hibernate e low fluorescence medium

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Hibernate E low fluorescence medium is a cell culture medium designed to maintain cells in a quiescent state with reduced metabolic activity. It is formulated to minimize the autofluorescence signal from the medium, making it suitable for applications that require low-background fluorescence detection.

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Lab products found in correlation

Lsm 880 airyscan confocal microscope, by Zeiss (2 mentions) Matlab, by MathWorks (2 mentions) Antimycin a, by Merck Group (1 mentions)

Close spelling variants of this product

Hibernate E (19 citations) Hibernate E medium (14 citations) Hibernate-E low fluorescence (4 citations) Hibernate medium (3 citations)

4 protocols using hibernate e low fluorescence medium

1

Imaging Neuronal ATP Levels and Mitochondrial Transport

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Transduced cortical neurons were transferred to chambers containing pre-warmed imaging buffer (Hibernate E low fluorescence medium (BrainBits) with 2% B27 and 0.5 mM GlutaMAX). A Zeiss LSM 880 Airyscan confocal microscope with a 40 × 1.3 NA oil immersion objective was used for live neuron imaging. To assess cellular ATP levels, images were collected at emissions 505–550 nm and above 545 nm within cell bodies or axons expressing GO-ATeam2 to measure ratiometric values which were generated in ImageJ (Huang et al. 2021 (link)). To analyze axonal mitochondrial transport, live images were captured along the microgrooves with 5-sec intervals, and kymographs were generated using ImageJ (Kang et al. 2008 (link)).
Huang N, & Sheng Z.H. (2022). Microfluidic devices as model platforms of CNS injury-ischemia to study axonal regeneration by regulating mitochondrial transport and bioenergetic metabolism. Cell Regeneration, 11, 33.
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2

Kymograph Analysis of Mitochondrial Dynamics

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Neurons on glass coverslips were placed in a 35-mm petridish containing the Hibernate E low-fluorescence medium (BrainBits) on a heated stage of 37 °C, and imaged with a 63×/N.A.0.9 water-immersion objective with excitation at 561 nm or 488 nm. We imaged axons positive for both mito-dsRed and EGFP which likely took up RNA duplex. Axons longer than 50 μm were selected for recording. Time-lapse movies were obtained continually with 5-s intervals for 3–5 min. For quantification, kymographs were generated from time-lapse movies by ImageJ, representing a 100-s period. Each kymograph was then imported into a macro written in Labview (NI, TX), and individual mito-dsRed puncta were traced using a mouse-driven cursor at the center of the mito-dsRed object. Using Matlab (The MathWorks, MA), we determined the following parameters: (1) the instantaneous velocity of each mitochondrion, (2) the average velocity of those mitochondria that are in motion, (3) the percent of time each mitochondrion is in motion, (4) stop frequency, and (5) turn back frequency.
Zhao Y., Song E., Wang W., Hsieh C.H., Wang X., Feng W., Wang X, & Shen K. (2021). Metaxins are core components of mitochondrial transport adaptor complexes. Nature Communications, 12, 83.
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3

Mitochondrial Dynamics in Neurons

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As described previously [40 (link)–42 (link)], neurons on glass coverslips were placed in a 35-mm petridish containing the Hibernate E low-fluorescence medium (BrainBits) on a heated stage of 37°C, and imaged with a 63×/N.A.0.9 water-immersion objective with excitation at 561 nm or 488 nm. Time-lapse movies were obtained continually with 3–5 sec intervals before and after Antimycin A (100 μM, Sigma-Aldrich) was added. Axons longer than 50 μm were selected for recording. Movie length ranged from 120 to 300 min. TMRM (T668, Molecular Probes) was applied at 250 nM for 10 min when needed. For quantification, kymographs were generated from time-lapse movies by ImageJ, representing a 100-sec period either right before or following different time points after addition of Antimycin A. Each kymograph was then imported into a macro written in Labview (NI, TX), and individual mito-dsRed puncta were traced using a mouse-driven cursor at the center of the mito-dsRed object. Using Matlab (The MathWorks, MA), we determined the following parameters: 1) the instantaneous velocity of each mitochondrion, 2) the average velocity of those mitochondria that are in motion, 3) the percent of time each mitochondrion is in motion, 4) stop frequency, and 5) turn back frequency. The intensity of mitochondria is measured using ImageJ.
Shaltouki A., Hsieh C.H., Kim M.J, & Wang X. (2018). Alpha-Synuclein Delays Mitophagy and Targeting Miro Rescues Neuron Loss in Parkinson’s Models. Acta neuropathologica, 136(4), 607-620.
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4

Axonal Transport Dynamics Imaging

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Transfected or infected neurons were transferred to chambers containing pre-warmed Hibernate E low fluorescence medium (BrainBits) supplemented with 2% B27 and 0.5 mM GlutaMAX. Live neurons were imaged with a 40x 1.3 NA oil immersion objective on a Zeiss LSM 880 Airyscan confocal microscope at 37°C. To image axonal mitochondrial transport, images were taken consecutively every 5 sec for a total duration of 7.5 min. To image axonal LAMP-1 transport, images were taken consecutively every 2 sec for a total duration of 3 min. Kymographs were generated using ImageJ (NIH) as previously described.19 (link) For each experimental group, all visible vesicles on kymographs were pooled together. Mitochondria were considered stationary if they remained immobile during the recording period; a mobile one was counted if the net displacement was > 5 μm. To measure axonal ATP levels, two emission images were collected at 505-550 nm and long pass above 545 nm along axons expressing GO-ATeam2, as described previously.9 (link) Ratiometric images were generated using ImageJ.
Huang N., Li S., Xie Y., Han Q., Xu X.M, & Sheng Z.H. (2021). Reprogramming an energetic AKT-PAK5 axis boosts axon energy supply and facilitates neuron survival and regeneration after injury and ischemia. Current biology : CB, 31(14), 3098-3114.e7.
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