Method Article

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

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DOI:

10.3791/60455

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December 12th, 2019

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1Institute for Frontier Life and Medical Sciences, Kyoto University, 2Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, 3Kyoto University Graduate School of Medicine, 4Kyoto University Graduate School of Biostudies

In This Article

Summary

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

Abstract

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Introduction

The mammalian brain is composed of a large number of various types of neurons and glial cells. All cells are generated from neural stem/progenitor cells (NPCs), which first proliferate to expand their numbers, then start to differentiate into neurons, and finally give rise to glial cells1,2,3,4,5. Once cells have differentiated into neurons, they cannot proliferate or increase their numbers, and, therefore, the maintenance of NPCs until later stages is important. Notch signaling via cell-cell interactions plays an important role in maintaining NPCs6,7. Notch ligands interact with the membrane protein, Notch, on the surface of neighboring cells and activates the Notch protein. After activation, proteolysis of Notch protein occurs, thereby releasing the intracellular domain of Notch (NICD) from the cell membrane into the nucleus8,9,10. In the nucleus, NICD binds to the promoter regions of Hes1 and Hes5 (Hes1/5) and activates the expression of these genes. Hes1/5 repress the expression of the proneural genes Ascl1 and Neurogenin1/2 (Neurog1/2)11,12,13,14. Because proneural genes induce neuronal differentiation, Hes1/5 play essential roles in maintaining NPCs. Furthermore, as proneural genes can activate the expression of the Notch ligand Delta-like1 (Dll1), Hes1/5 also repress the expression of Dll1. Therefore, the expression of Dll1 leads to neighboring cells being negative for Dll1 via Notch signaling. In this way, cells inhibit adjacent cells from following their same fate, a phenomenon known as the lateral inhibition8. In the developing brain, lateral inhibition plays a role in generating various different cell types.

Real-time imaging at the single cell level reveals dynamic expressions of the components of Notch signaling in NPCs15,16,17. Notch signaling activates the expression of Hes1, but Hes1 protein binds to its own promoter and represses its own expression. Furthermore, Hes1 is an extremely unstable protein, that is degraded by the ubiquitin-proteasome pathway; therefore, the repression of its own promoter is only short lived and then the transcription starts again. In this way, the expression of Hes1 oscillates at both the transcription and translational levels in a 2 h cycle18. The oscillatory expression of Hes1, in turn, induces the oscillatory expression of the downstream target genes, such as Ascl1, Neurog2, and Dll1, via periodic repression15,16,17,19. While proneural genes can induce neuronal differentiation, their oscillatory expression is not sufficient for neuronal differentiation; rather their sustained expression is essential for the neuronal differentiation. The oscillatory expression of proneural genes is important for maintaining NPCs rather than for inducing neuronal differentiation14,15,16. The expression of Dll1 oscillates at both the transcription and translational levels during various morphogenesis, such as neurogenesis and somitogenesis. The dynamic expression of Dll1 is important for the normal morphogenesis and steady expression of Dll1 induces defects in neurogenesis and somitogenesis17. These findings demonstrate the important function that the dynamics of gene expression and protein kinetics have on the regulation of various developmental events (i.e., different expression dynamics produce different outputs in cellular behaviors).

To analyze the dynamics of Notch signaling, the static analysis of tissues and cells are insufficient because they are constantly changing. Real-time imaging of single cells is a powerful tool to reveal the dynamics in gene expression. The dynamic expression of Notch signaling molecules undergo rapid cyclic responses in the period of 2-3 h. This rapid periodic expression presents two difficult problems for the real-time monitoring: (1) the expression of the molecules is suppressed to low levels, and (2) rapid turnover requires fast-response reporters. To overcome these problems, we previously developed a bioluminescence real-time imaging method20. Because the bioluminescence reporter has a higher sensitivity and shorter maturation time than fluorescent reporters, this strategy enables us to monitor the rapid dynamics in living cells. Using real-time visualization, we found that more genes exhibited dynamic expression than we had previously thought. In addition, the number of reports showing expression and protein dynamics in living cells and the significance of these dynamics in various biological events has increased, suggesting a fundamental role of the dynamics in gene expressions21,22.

In this report, we describe a way to visualize the expression of the Notch ligand Dll1 in NPCs in both dissociated cultures and in cortical slice cultures. To monitor the dynamics of Dll1 transcription at single cell levels, we generated dissociated cultures of NPCs derived from the embryonic telencephalon of transgenic mice carrying pDll1-Ub-Fluc reporter, a Dll1 promoter-driven destabilized luciferase reporter. To monitor Dll1 protein dynamics in vivo, we introduced the Dll1-Fluc fusion reporter into NPCs in the cortex and visualized the expression of the reporter in NPCs in cortical slice cultures. Real-time imaging enabled us to capture the various features of gene expression and protein dynamics in living cells at high temporal resolution.

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Protocol

All the procedure including animal subjects have been approved by Institutional Animal Care and Use Committee at the institute for Frontier Life and Medical Sciences, Kyoto University.

1. Bioluminescence reporters

NOTE: The luciferase reporter is suitable for measuring the rapid dynamics of promoter activity by fusing the degradation signal. Moreover, the luciferase fusion reporter enables monitoring of the protein dynamics in the single cell. Both types of reporters are available for mono-layer culture (dissociation culture) and tissue culture (slice culture) experiment.

  1. Reporter for monitoring Dll1 promoter activity17
    NOTE: To monitor the rapid changes in gene expression, the rapid-response and unstable reporter is essential. Firefly luciferase (Fluc) shows rapid maturation compared with fluorescein reporters. Because ubiquitin fused luciferase reporter (Ub-Fluc) shows rapid degradation and rapid-turnover, it is very useful to monitor the dynamic gene expression in living cells20.
    1. Use Dll1 promoter-driven destabilized luciferase reporter, ubiquitinated luciferase (pDll1-Ub-Fluc, Figure 1A upper panel), to monitor rapid changes in Dll1 expression at a transcriptional level17.
    2. Generate a transgenic mouse line carrying pDll1-Ub-Fluc for the stable expression of this reporter.
  2. Reporter for monitoring Dll1 protein dynamics17.
    1. For generation of knock-in mice, insert the luciferase cDNA into the 3´ termini of the Dll1 coding region so that Dll1-luciferase fusion protein is expressed (Dll1-Fluc reporter, Figure 1A, lower panel)17.
    2. Monitor the expression dynamics of Dll1 at protein levels by measuring the luciferase activity.
    3. For monitoring Dll1 protein dynamics at tissue levels, introduce the Dll1-Fluc reporter into NPCs in the embryonic telencephalon by in utero electroporation.

2. Bioluminescence imaging system

  1. Construct a bioluminescence live-imaging system using an inverted microscope installed with a highly sensitivity, water-cooled CCD camera. In order to reduce the noise and obtain high sensitivity, cool the camera with chilled water provided by the water circulator at an optimized temperature of -90 °C.
  2. For the live cell/tissue imaging, install the incubation system, controlling the temperature and mixed gas, to the microscope stage.
  3. For imaging the expression of destabilized luciferase reporter, use high numerical angle (NA: 1.30) 40x oil-immersion objective lens. The working distance of this lens is 0.2 mm, it is available for not only mono-layer cell culture but also slice culture.
  4. For dual monitoring of luciferase and fluorescein reporter expression, install the LED illumination device to the light path of the microscope.
  5. Control the time-lapse imaging with a software associated with the microscope (e.g., Multidimensional acquisition program of MetaMorph software).
  6. Prepare the entire system in a dark room, because the signal of the destabilized luciferase reporter is very faint, and to avoid the extraneous light prevent the acquisition.

3. Neural Stem/Progenitor Cell (NPC) dissociation cultures

  1. Preparation of culture media, dishes, and reagents for NPC dissociation culture
    1. Prepare N2/B27 media for culturing neural stem/progenitor cells with a final concentration of 1x N2, 1x B27, 1 mM N-acetylcysteine, 10 ng/mL bFGF and 50 U/mL Penicillin/Streptomycin in DMEM/F12 media.
    2. Prepare papain solution for dissociation containing 7 U/mL papain, 0.006% DNase and 1 mM N-acetylcysteine in EBSS media.
    3. Prepare 100 mM of luciferin sodium solution for luminescence imaging. Dilute 100 mM luciferin solution in N2/B27 media to a final concentration of 1 mM.
      NOTE: Luciferin shows auto-fluorescein itself. In the case of luciferase-fluorescence dual imaging, especially EGFP, lowering the concentration of luciferin to 0.5 mM is better.
    4. Coat a 35-mm glass bottom dishes (glass diameter 27-mm) with 40 μg/mL of poly-L-lysine (PLL) solution for 1 h at room temperature. After incubation, wash the plates 3x with PBS and let it dry.
  2. Dissection of embryos and dissociation of NPCs
    1. After euthanizing a pregnant pDll1-Ub-luc transgenic mouse (embryonic day 12.5 (E12.5)) by CO2 asphyxiation and cervical dislocation, cut through the abdomen and take out the uterus.
      NOTE: Researchers must follow the regulations and guidelines of animal research committee of their institution. If anesthesia or pain relief drugs are used, use them properly.
    2. Transfer the uterus in a 10 cm Petri dish containing 25 mL of ice-cold PBS. Take out the embryos from the uterus in ice-cold PBS, using micro scissors and fine forceps.
    3. Cut off the head of each embryo using scissors and transfer it to 10 cm Petri dishes containing ice-cold DMEM/F12. Remove the epidermis and cartilage surrounding the brain and transfer the brain to 35 mm Petri dish containing ice-cold 3 mL of N2/B27 media.
    4. Cut off the right and left telencephalon from diencephalon (Figure 1Ba-f, C,D). Remove the meninges covering the surface of telencephalon using fine forceps (Figure 1Bi). Using fine forceps again, take out the dorso-lateral part of cortex from the telencephalon (Figure 1Bg,h,j-l E).
    5. Transfer the tissues to new 1.5 mL tubes using a P1000 pipette and remove any extra medium with a P200 pipette. Add 0.1 mL of papain solution per brain tissue (a pair of the cortex). After 15 min of incubation at 24 °C, gently pipette the samples 10 times with P1000 pipette. Incubate the samples again for 15 min at 24 °C.
    6. Gently pipette the samples 10 times with P1000 pipette. Centrifuge the samples for 3 min at 400 x g and room temperature (RT). Discard the supernatant.
    7. Add 1 mL of DMEM/F12 media to the pellet. Gently pipette 10 times with P1000 pipette. Centrifuge the samples for 3 min at 400 x g and RT. Discard the supernatant. Repeat this at least 2 more times.
    8. To the pellet, add 0.5 mL of N2/B27 media containing 1 mM luciferin and mix well. Seed the cells (1 x 106 cells) to a PLL-coated glass bottom dish. Culture the cells in the CO2 incubator for 1 h. Once the cells adhere to the dish, add 2 mL of N2/B27 media, containing 1 mM luciferin.
  3. Visualization of luciferase reporter expression in NPC dissociation culture
    1. Start up the luminescence live-imaging system before performing the dissection. For NPC culture, set the temperature of the stage incubator at 37 °C and set the setting of the gas mixture 20% O2, 5% CO2.
    2. Put the immersion oil to the objective lens. Place the sample dish on the microscope stage. View the field manually and choose the best position and the focus of the cells of interest. Click live to acquire the test image.
    3. Run the time-lapse acquisition by 2-dimensional (luminescence and bright field) acquisitions for 24 h with Multi-Dimensional Acquisition program. Choose Multi-Dimensional Acquisition program and set the acquisition setting as follows (step 3.3.6).
    4. For luminescence image acquisition, use the following camera settings: low-transfer rate (50 kHz), 2 x 2/4 x 4 binning, 10 min/5 min exposure time. For bright-field image acquisition, use the following settings: intermediate-transfer rate (1 MHz), 1 x 1 binning and 100 ms exposure time.

4. In utero electroporation

NOTE: This is performed for the introduction of Dll1-Fluc reporter into the neural progenitor cells.

  1. Preparation of tools and reagents for electroporation
    1. Prepare micro capillaries for injection of DNA into the ventricle of the embryonic telencephalon. Stretch a grass capillary with heat and cut and polish the tip of it with the polishing machine.
    2. Prepare a mixture of DNA with dye (e.g., Trypan blue). The concentration of each DNA is 1 μg/μL in PBS and add the dye to a final concentration of 10%.
      NOTE: Use the mixture of DNA for visualization of Dll1 protein dynamics as follows: Dll1-Fluc (Dll1 protein reporter, Figure 2E upper) and pEF-EGFP (EF promoter driven EGFP expression vector, Figure 2E lower), monitoring the location and morphology of transfected cells.
    3. Prepare two types of anesthetics: 3.88 mg/mL of pentobarbital and 0.12% of xylazine, in sterile 1x PBS.
    4. Use the sterile instruments and surgical gloves during operation.
  2. In utero electroporation
    1. Anesthetize a time pregnant ICR mouse (E12.5) by intraperitoneal administration of anesthetics with 500 μL of 3.88 mg/mL pentobarbital and 500 μL of 0.12% xylazine.
      NOTE: Researchers must follow the regulation of animal experiments. If one needs to use anesthesia or pain relief drugs, use them properly.
    2. Clip the hair and disinfect the skin with surgical scrub.
    3. Check for the lack of toe pinch response. Once pedal reflex is not observed, make a 2-3 cm cut through the abdomen along the midline. Put gauzes around the dissected part and wet the gauzes with warm PBS not to dry up the incision. Take out the right uterine horn gently with ring-shaped forceps and count the number of embryos.
    4. Inject 1-2 μL of the mixed DNA into the ventricle of the telencephalon of embryos gently with micro capillary.
      NOTE: When the injection is successful, one can see the blue color of the dye over the surface of the uterus.
    5. Wet the uterus and the electrode before providing the voltage pulses, and then hold the head of an embryo gently. Set the positively charged electrode to the side of the hemisphere, in which the DNA was injected. Ensure that the condition of the pulse of electroporation is 30-50 V for 50 ms, the interval of the pulses is 1 s (50 ms charge and 950 ms non-charge). Provide 5 pulses to one embryo and check that bubbles are generated from the negatively charged electrode.
      NOTE: Make sure the direction of the electrode: DNA is incorporated into cells on the side of positive electrode. During this procedure, do not move the position of the electrode.
    6. Repeat the steps 4.2.3-4.2.4 for other embryos and return the uterine horn to the abdominal cavity. Carry out the same procedure to the embryos in the left uterine horn.
    7. After putting back the left side, suture the incision by a silk suture (4-0) with a needle (17 mm). Before closing the incision completely, put warm PBS into the abdominal cavity
      NOTE: The interval between sutures is around 2 mm.
    8. After finishing the surgery, place the mouse on a heating pad for post anesthesia recovery. House the mouse individually.
      NOTE: Researchers must follow their local instiutional regulation of animal experiments. If one needs to use pain relief drugs, use them properly.

5. Preparation of slice cultures of the developing cortex and visualization of luciferase reporter expression in the cortical slices

  1. Preparation of culture media and reagents for slice cultures of the cortex
    1. Prepare enriched media for culturing cortical slices containing 5% fetal bovine serum and 5% horse serum in N2/B27 media.
    2. Bubble 100% O2 gas through DMEM/F12 media for dissection and making cortical slices.
      NOTE: To use oxygen gas safely, store the O2 cylinder in the cylinder cabinet and monitor the oxygen concentration in the air by an oxygen concentration meter.
    3. Dilute 100 mM luciferin sodium solution in enriched media to a final concentration of 1 mM.
      NOTE: Luciferin shows auto-fluorescein itself. In the case of luciferase-fluorescence dual imaging, especially EGFP, lower concentration of luciferin (0.5 mM) is better.
    4. Set the multi-gas incubator at 37 °C by 40% O2 and 5% CO2.
  2. Dissection of embryos and examination of the expression of fluorescent reporter
    1. After euthanizing the pregnant mouse to which the reporters (E13.5) are introduced by in utero electroporation (step 4.2), cut through the abdomen and take out the uterus.
      NOTE: Researchers must follow the regulation of animal experiments. If using anesthesia or pain relief drugs, use them properly.
    2. Transfer the uterus to a 10 cm Petri dish containing PBS. Take out the embryos from uterus in PBS, using micro scissors and fine forceps. Cut off the head of each embryo and transfer to a 10 cm Petri dish containing DMEM/F12 media bubbled with 100% O2 gas. Remove epidermis and cartilage surrounding the brain in DMEM/F12 media.
    3. Put the brain on the lid of the Petri dish with ring-shaped forceps and set the lid on the fluorescence stereoscopic microscope stage. Check the region of the cortex expressing fluorescent protein under the excitation light (Figure 2A,B).
    4. Transfer the brain to a silicone rubber cutting board filled with 30 mL of DMEM/F12 media bubbled with 100% O2 gas. Remove meninges covering the surface of the telencephalon by fine forceps.
    5. Cut the border between medial and lateral part of the dorsal telencephalon and separate into two hemispheres using micro surgical knife or fine forceps. Using a micro surgical knife, cut the cortex like stripes and make cortical slices (Figure 2C,D). Pipette slices with medium using pipette and transfer them to enriched media in a 35 mm dish.
    6. Put the slices on the culture inserts in the glass bottom dishes with enriched media. Correct the direction of slices using fine forceps. Set up the cut surface of the slices to the surface of culture insert. Remove the extra media by a pipette. Incubate the slices in multi-gas incubator set by 40% O2 and 5% CO2 at 37 °C for 30 min.
    7. Add 300 μL of enriched media containing 1 mM luciferin to the outside of culture insert in the glass bottom dish.
  3. Visualization of luciferase reporter expression in slice cultures
    1. Start the luminescence live-imaging system before starting the dissection. Use the following condition of cortical slice cultures: 40% O2, 5% CO2 and 37 °C.
    2. Put the immersion oil to 40x objective lens. Put the sample dish to the stage of microscope. Acquire the test image of fluorescent and set the position and the focus plane to the region of interest under the illumination of excitation light.
    3. Run the time-lapse acquisition by 3-dimentional (luminescence, fluorescence and bright field) acquisitions for 24 h (Figure 2F-H). For luminescence image acquisition, use the following camera settings: low-transfer rate (50 kHz), 2x2/4x4 binning, 10 min/5 min exposure time. For the fluorescence and bright field image acquisition, use the following settings: intermediate-transfer rate (1 MHz), 1x1 binning and 100 ms exposure time.

6. Image processing and analysis

  1. Connect each image and make the stack images. Click Import Image Sequence from the File menu (File | Import | Image Sequence) and choose the folder, where the acquired images are saved. Put some words contained in file name to the column of File name contains.
  2. To remove the noise of cosmic ray on the bioluminescence images, apply the SpikeNoise Filter plug-in of ImageJ/Fiji. Open the stack images and click SpikeNoise Filter.
  3. Apply Savitzky Golay Temporal Filter plug-in to get clear dynamics of reporter expression. Open the stack images and click Savitzky Golay Temporal Filter.
  4. To measure the intensity of reporter expression, apply Z Axis Profile Plus plug-in to each single cell. Select the cells and set ROIs, region of interest and click Z Axis Profile Plus.

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Results

Expressions of the genes Hes1/7 exhibit 2 h oscillation cycle in various cell lines and during somitogenesis. Furthermore, the period of oscillation is very short and both their mRNAs and proteins are extremely unstable with the half-lives of around 20 min. If using a slow response reporter, we cannot trace such rapid dynamics, and if using a stable reporter, it gradually accumulates while the gene expression oscillates. Thus, the reporter must be rapidly degraded to monitor the rapid turnover of such cyclically...

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Discussion

The components of Notch signaling show oscillatory expressions in synchrony during somitogenesis but out of synchrony during neurogenesis, leading to the difficulties in capturing the expression dynamics by static analysis in the latter case. Thus, real-time monitoring is required to reveal the expression dynamics of Notch signaling components, such as Hes1 and Dll1. Because the periods of the expressions of Hes1 and Dll1 oscillations are extremely short, approximately 2-3 h, rapid res...

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Disclosures

The authors have no conflicting financial interest.

Acknowledgements

We thank Yumiko Iwamoto for supporting the production of the video. We are also grateful to Akihiro Isomura for discussion and supports of image analysis, Hitoshi Miyachi for technical supports for generation of transgenic animals, Yuji Shinjo (Olympus Medical Science), Masatoshi Egawa (Olympus Medical Science), Takuya Ishizu (Olympus Medical Science) and Ouin Kunitaki (Andor Japan) for the technical support and discussions of the bioluminescence imaging system. This work was supported by Core Research for Evolutional Science and Technology (JPMJCR12W2) (R.K.), Grant-in-Aid for Scientific Research on Innovative Areas (MEXT 24116705 for H.S. and MEXT 16H06480 for R.K.), Grant-in-Aid for Scientific Research (C) (JSPS 18K06254) (H.S.), Takeda Foundation (R.K. and H.S.), and Platform for Dynamic Approaches to Living System from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Bioluminescence Imaging System
Chilled water circulator (chiller)JulaboModel: F12-ED
Cooled CCD cameraAndor TechnologyModel: iKon-M 934
Incubator systemTOKAI HITModel: INU-ONICS
Inverted microscopeOlympusModel: IX81
Inverted microscopeOlympusModel: IX83
LED illumination deviceCoolLEDModel: pE1
MetaMorphMOLECULAR DEVICESModel: 40000
Mix gas controllerTokkenModel: TK-MIGM OLO2
Objective lensOlympusModel: UPLFLN 40X O
Preparations for Dissection
Dissection microscopeNikonModel: SMZ-2B
Fluorescence stereoscopic microscopeLeicaModel: MZ16FA
Fine forcepsDUMONTINOX No.5
Scissors, Micro scissors
Forceps
Ring-shaped forceps
10-cm plastic petri dishgreiner664160-013
35-mm plastic petri dishgreiner627160
PBSNacalai Tesque14249-24
DMEM/F12invitrogen11039-021
Reagents for NPC dissociation culture
B27 supplementinvitrogen12587-010
bFGFinvitrogen13256-029Stock solution: 1 μg/ml in 0.1% BSA/PBS
D-luciferinNacalai Tesque01493-85Stock solution: 100mM in 0.9% saline
DNaseWorthington Biochemical CorporationLK003172Stock solution: 1000U/ml in EBSS
EBSSWorthington Biochemical CorporationLK003188
Glass bottom dishIWAKI3910-035
N2 supplement (100x)invitrogen17502-048
N-acetyl-cysteinSigmaA-9165-25G
PapainWorthington Biochemical CorporationLK003178Stock solution: 7U/ml in EBSS
Penicillin/StreptmycineNacalai Tesque09367-34
Poly-L-lysineSigmaP-628140 mg/ml in DW
Preparations for in utero electroporation
50-ml syringeTERUMO181228T
ElectrodeNeppagene7-mm
ElectroporatorNeppageneCUY21 EDIT
Forceps
GauzesKawamoto co.7161
Micro capillaryMade in-house
PBSNacalai Tesque14249-24
PentbarbitalKyoritsuseiyakuSomnopentyl
Ring-shaped forceps
Scissors, Micro scissors
Suture needleAkiyama MEDICAL MFG. COF17-40B2
XylazineBayerSeractal
Preparations for Slice culture
10-cm plastic petri dishgreiner664160-013
35-mm plastic petri dishgreiner627160
Culture insertMilliporePICM01250
DMEM/F12invitrogen11039-021
Fetal Bovine SerumSigma172012-500ML
Fine forcepsDUMONTINOX No.5
Forceps
Horse SerumGibco16050-122
Micro surgical knifeAlcon19 Gauge V-Lance
Multi-gas incubatorPanasonicMCO-5MUV-PJ
N2/B27 mediaMade in-houseref. NPC dissociatioin culture
PBSNacalai Tesque14249-24
Ring-shaped forceps
Scissors, Micro scissors
Silicon rubber cutting boardMade in-house

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Neural Stem CellsIn Utero ElectroporationLuciferase ReporterProtein DynamicsGene Expression MonitoringLive Cell ImagingCortical Slice CultureSingle Cell Resolution

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