Commercially available hiPSC-neurons were used in this study [iCell Neuron: Cellular Dynamics International (CDI), Madison, WI, USA] and were cultured according to the manufacturer’s instructions. The cells were plated in 8-well glass chambers (155409JP, Nunc, Waltham, MA, USA), 3.5-cm culture dishes, or 96-well plastic plates, which were pre-coated with poly-D -lysine (50 μg/ml for 24 h, P6407, Sigma, Darmstadt, Land Hessen, Germany) followed by a top coating with laminin (5 μg/ml for 2 h, L2020, Sigma, Darmstadt, Land Hessen, Germany), at a density of 75,000 cells/cm2 and cultured in iCell Neuron-maintenance medium (CDI). The medium was changed 24 h after plating and after every 3 days. The reproducibility of all experimental data was confirmed by three independent experiments using iCell Neurons with different lot numbers (#139991, #1361234, #1360614). The all experiments using hiPSC-neurons were approved by the Research Ethics Committee of National Institute of Health Sciences (NIHS) in accordance with the Declaration of Helsinki.
P6407
Manufactured by Merck Group
Sourced in Germany, United States
The P6407 is a laboratory equipment product from Merck Group. It is a precision device designed for scientific research and analysis applications. The core function of the P6407 is to provide accurate and reliable measurements, though the specific details of its intended use are not provided.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
L2020, by Merck Group (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Neurobasal medium, by Thermo Fisher Scientific (3 mentions)
P6282, by Merck Group (2 mentions)
Lk003176, by Worthington (2 mentions)
A9647, by Merck Group (2 mentions)
P35g 1.5 14 c, by Merck Group (2 mentions)
N1408, by Merck Group (2 mentions)
F7524, by Merck Group (2 mentions)
Glutamine, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Lsm 880, by Zeiss (2 mentions)
4 2 hydroxyethyl 1 piperazineethanesulfonic acid, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Harvard Bioscience (1 mentions)
L11 044, by Harvard Bioscience (1 mentions)
N2513, by Merck Group (1 mentions)
Lipofectamine ltx reagent, by Thermo Fisher Scientific (1 mentions)
33 protocols using p6407
1
Culturing hiPSC-Derived Neurons
2
Scaffold Fabrication for bTEBV
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
It is crucial that a scaffold used to fabricate bTEBV provides a suitable template for the cells to adhere to [59 ]. In our studies we added to the basic scaffold the following components at a concentration of 3 μg/ml: poly(l -Lys) (as hydrobromide; mol wt 70,000–150,000; P6282, Sigma), poly(d -Lys) (as hydrobromide; mol wt 70,000–150,000; P6407, Sigma) or His/Gly-tagged RGD. Poly(l -Lys) and poly(d -Lys) were added to the scaffold after hardening with CaCl2, while His/Gly-tagged RGD was added prior to the crosslinking of the basic scaffold components (N,O-CMC, polyP, alginate and gelatin). Subsequently the material was extruded or printed and finally hardened as described. The material is termed “poly(l -Lys)-scaffold”, “poly(d -Lys)-scaffold” or “RGD-scaffold”.
3
Imaging Hippocampal Neurons Expressing Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sterilized glass coverslips were functionalized with poly-d -lysine in hydrobromide solution (70 μg/ml; P6407; Sigma-Aldrich, St. Louis, MO) for 1 h and rinsed off with double-distilled H2O. Mouse hippocampal neurons were prepared as previously described (Wienisch and Klingauf, 2006 (link)). In brief, primary neurons prepared from embryonic day 18 mice were plated onto coverslips using Neurobasal Medium, 21103-049; Life Technologies), supplemented with B27 (17504-044; Life Technologies), penicillin/streptomycin (12212; Biochrom AG) and 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (15630; Life Technologies). Transfection was accomplished with Lipofectamine 2000 (11668-027; Life Technologies) following the manufacturer’s protocol. After 14–24 h of expression, cells were imaged each 10 s for 10 min at 37°C. All animal protocols were approved by the Veterinär- und Lebensmittelüberwachungsamt Münster.
4
Differentiation of PC12 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Glass coverslips were coated with 50 μl of poly-d -lysine in hydrobromide solution (P6407; Sigma-Aldrich) and incubated at 37°C for 30 min. After incubation, coverslips were washed twice with sterile water and again incubated with 100 μl of collagen solution (in 20 mM acetic acid) at 37°C for 45 min. Next coverslips were washed twice with phosphate-buffered saline (PBS; 10010-023; Life Technologies). PC12 cells were cultured on coated coverslips using DMEM containing 4.5 g/l d -glucose (31053-028; Life Technologies), 10% fetal bovine serum (FBS: L11-044; Biochrom AG), 5% horse serum (B15-023; PAA, GE Lifesciences, Fairfield, CT), 1% Glutamax (35050-061; Life Technologies) and 1% penicillin/streptomycin (10.00 U/ml; 12212; Biochrom AG) and incubated at 37°C and 5% CO2. Transfection was accomplished 1 d after plating with Lipofectamine 2000 (11668-027; Life Technologies) following manufacturer’s protocol. At 24 h after transfection, culture medium was exchanged with differentiation medium containing DMEM with 4.5 g/l d -glucose (31053-028Life Technologies), 0.67% FBS (Biochrom AG, L11-044), 0.33% horse serum (B15-023; PAA), 1% GlutaMax, 1% penicillin/streptomycin (10,00 U/ml; 12212; Biochrom AG), and 1 μl of rat nerve growth factor-β (N2513; Sigma-Aldrich). The differentiating PC12 cells were imaged 5 d after plating at 10-s intervals.
5
Calcium Imaging of HEK Cells Expressing αvβ3 Integrin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK cells expressing human αvß3 integrin72 (link) were cultured in DMEM supplemented with 10% FBS and 20 mM glutamine in a 5% CO2 incubator. A stable calcium reporter line was generated with a GCaMP6f lentivirus (Cellomics Technology PLV-10181-50) followed by FACS sorting. For screening experiments and characterization of each candidate channel, GCaMP6f-expressing HEK cells were seeded on 12-well cell culture plates with 18 mm glass coverslips coated with PDL (10 μg/μl; Sigma-Aldrich P6407) for 1-2 h. Coverslips were washed with Milli-Q water and cells seeded at a density of 250000 cells/well. Cells were transfected with Lipofectamine LTX Reagent (ThermoFisher 15338100) according to the manufacturer’s protocol and 24 h after plating, using 500 ng DNA of the clone of interest for each well. Cells were kept at 37 °C for an additional 24 h before imaging on our ultrasound stimulation setup.
6
Preparation of Hippocampal Neuron Cultures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Hippocampal cultures were prepared from E18 time-pregnant mice as previously described (Gu et al., 2016a (link)). Briefly, the mouse hippocampi were dissected out in ice-cold Hank’s balanced salt solution, and digested with papain (Worthington, LK003176) solution at 37°C for 45 min. After centrifugation for 5 min at 800 rpm, the pellet was resuspended in DNase I-containing Hank’s solution, then was mechanically dissociated into single cells by gentle trituration using a pipette. Cells were placed on top of Hank’s solution mixed with trypsin inhibitor (10 mg/ml, Sigma T9253) and BSA (10 mg/ml, Sigma A9647), and centrifuged at 800 rpm for 10 min. The pellet was resuspended in Neurobasal plating media containing 2% B27 supplements and L-glutamine (2 mM). Neurons were plated at a density of 150,000–200,000 cells/well on poly-D-lysine (Sigma P6407)-coated 12 mm glass coverslips residing in 24-well plates for electrophysiology recording, and a lower plating density (100,000–150,000 cells/well) was adopted when neurons were used for immunocytochemistry. Culture media were changed by half volume with Neurobasal maintenance media containing 2% B27 supplements and L-glutamine (2 mM) twice a week.
7
Metabolic Flux Analysis with Seahorse XF
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Seahorse XF 96 Metabolic Flux Analyzer (Seahorse Bioscience, Lexington, MA, USA) was used to measure the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Cells entered the analysis after culturing as indicated. After harvesting, the cells were resuspended in prewarmed Seahorse medium (Agilent Seahorse XF RPMI Medium pH 7.4, supplemented with 10 mM Seahorse XF d -Glucose and Seahorse XF 2 mM l -Glutamine). Cells were counted by the Countess Automated Cell Counter (Thermo Fisher) and 1.1 × 105 viable cells per well (6 technical replicates per condition) were plated into a poly-d -lysine (P6407, Sigma-Aldrich) coated Agilent Seahorse XF96 Cell Culture Microplate (Seahorse Bioscience). To immobilize the cells on the bottom, the plate was centrifuged at 500× g for 5 min. Subsequently, the plate was put in a non-CO2 incubator at 37 °C for 30 min, 130 µL of Seahorse medium were added to each well and the plates were returned for another 25 min to the non-CO2 incubator at 37 °C until analysis. OCR and ECAR were measured five times with 30 s intervals of mixing.
8
Quantifying DRG Neurite Length with Phentolamine
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Based on the protocol described previously8 (link). 4–6-week-old C57BL/6J mice dissociated DRG neurons were plated onto the poly-D-lysine (100 µg/mL), laminin (10 µg/mL) and the aggrecan (50 µg/mL) (P-6407, L-2020, A-1960, Sigma) coated coverslips in the 24-well culture plates and cultured in Neurobasal medium (1088802; Thermo Fisher Scientific) with B27 supplement containing penicillin, streptomycin, 1 mM l -glutamine, 50 ng/mL NGF, 2 ng/mL GDNF, and 10 mM AraC at 37°. For drug treatment, DRG neurons were cultured for 72 h in the presence of the drug phentolamine at 1, 3, 5, 8, 10, 12, 20 μm concentrations. The cells were post-fixed 4% paraformaldehyde (PFA) followed by phosphate buffer saline (PBS) washing and immunostained with anti-mouse b-III-tubulin (1:1000; 801201, BioLegend). Coverslips were then inverted and mounted on the glass-slides using Prolong Gold antifade reagent with DAPI (P36935, Fisher Scientific). Images were captured covering the entire coverslip area at × 20 magnification using an Olympus Fluorescent Microscope. Total neurite length was quantified with an ImageJ plug-in, NeurphologyJ38 (link) (RRID: SCR_003070). Neurphology J operates on the entire image and quantifies the neurite length in pixels. Data were obtained from at least 4 separate experiments repeated in duplicates.
9
Preparing Tissue and Organoid Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Prior to mounting, glass-bottomed 24-well plates (Cellvis P24-1.5H-N) were coated with 1 mg/ml PDL (Sigma-Aldrich P6407). 75 μl of the PDL solution was transferred into the center of each well and brushed to the edges with a fine paintbrush to ensure total coverage. The plates were rocked on a shaker for 5 min, after which the PDL was collected for reuse. Wells were rinsed three times with deionized water and let dry for at least 2 hr. Tissue sections were washed three times in PBS, loaded into wells containing 500 µl 1× PBS, which was subsequently aspirated allowing the tissue to lie flat and dry for approximately 20 min until there was no visible liquid remaining around the edges of the sections. The tissue was then rinsed for 30 s with 4% PFA and washed in PBS three times for 5 min each. Organoids sections were gently aspirated using a P1000 pipet with a trimmed tip and transferred into a well of a glass bottomed well plate. The sections were allowed to settle onto the glass before carefully removing the excess liquid and allowing to dry. Mounted organoid sections were additionally fixed with 4% PFA for 15 min followed by three washes with PBS. Prior to staining, the organoid sections were washed with the elution buffer three times for 5 min as a form of mild antigen retrieval.
10
HEK293 Cell Imaging with Poly-D-Lysine
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Glass bottom cell culture plates (MatTek, P35G-1.5-14-C) were coated with poly-D-lysine (50 ng/ml, Sigma, P6407) for 1–2 h at 37°C, after which the poly-D-lysine was removed, and coverslips were washed 3 X with sterile water and one time with DMEM. HEK293 cells were detached and resuspended in supplemented DMEM as described above, counted using a hemocytometer, plated (250,000 cells per well) and were transfected after 24 h with 0.75 g DNA. Twenty-four h after transfection, media was replaced by serum free DMEM and cells were incubated at 37°C and 5% CO2 for 1–2 h. To image, the serum free DMEM was replaced by phosphate-buffered saline (room temperature). The plates were transferred to the microscope (LeicaSP8X) and cells were imaged using a 40X oil objective with the following settings: 485 excitation and 525 emission wavelength, 5% laser power, HyD hybrid detector and a scan speed of 200 lines Hz (0.388 frames per second) with bidirectional scanning. All agents were prepared in PBS supplemented with 1 mg/ml BSA and spiked into buffer of cell on the microscope stage (Final concentration BSA = 0.1 mg/ml).
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!