Cells of passage 3–12 were washed with PBS (Life Technologies), centrifuged down (300xg for 5 min), resuspended in PBS, and placed on AdcellTM diagnostic slides (Thermo Fisher Scientific). Single cells were isolated under the microscope in 1 µl PBS using a micromanipulator (Patchman NP2) with pump (CellTram, both Eppendorf) and placed into 2 µl of lysis buffer (0.2% Triton X-100 [Sigma-Aldrich] and 4 U recombinant RNase inhibitor [Clontech Takara]). Samples were stored at −80 °C for up to 6 months.
Patchman np2
Manufactured by Eppendorf
Sourced in Germany
The Patchman NP2 is a compact and user-friendly micromanipulator designed for patch-clamp electrophysiology experiments. It provides precise and stable positioning of microelectrodes or pipettes during intracellular recordings and cell injections.
Automatically generated - may contain errors
Lab products found in correlation
Celltram, by Eppendorf (2 mentions)
Triton x 100, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Recombinant rnase inhibitor, by Takara Bio (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Rhs2000 stimulation recording controller, by Intan Technologies (1 mentions)
Rhs2116 amplifier chips, by Intan Technologies (1 mentions)
Buzsaki 32 a32, by NeuroNexus (1 mentions)
Kapa library quantification kit, by Roche (1 mentions)
Nextera xt, by Illumina (1 mentions)
Femtojet, by Eppendorf (1 mentions)
Transferman nk2, by Eppendorf (1 mentions)
Axio vert a1 inverted microscope, by Zeiss (1 mentions)
Piezoxpert, by Eppendorf (1 mentions)
Femtojet express, by Eppendorf (1 mentions)
6 protocols using patchman np2
1
Single-Cell Isolation and Lysis
2
Transient Transfection of HEK293 Cells
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Transient transfection of HEK 293 cells with plasmid pcDNA6.2-hERG (Y652A) was performed with the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. Transfected cells were cultivated in 6-well plates using 2 ml medium per well.
Selection of the stably transfected and fluorescent cells was performed using the Eppendorf micromanipulator PatchMan NP2.
Selection of the stably transfected and fluorescent cells was performed using the Eppendorf micromanipulator PatchMan NP2.
3
Extracellular Recording of Spinal Cord Neurons
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For the recording of action potentials, we used microelectrode arrays (MEAs) consisting of 32-recording sites distributed across 4 shanks separated by 200 μm (Buzsaki 32-A32, NeuroNexus). With the electrode mounted on a computer-controlled micromanipulator (Patchman NP2, Eppendorf) and placed by the side of the preparation, the level of the Sylgard floor was taken as zero reference. Then we placed the MEA over the L3–L4 border and lowered the electrode deep in the preparation until the last row of sensors was 100 μm below the sectioned border. Then, the electrode position was adjusted with smooth up and down movements to maximize the number of spontaneously active neurons recorded and allowed to stabilize for 30 min prior to start the experiment.
Signals from the MEA were amplified, band pass filtered between 200 and 3 KHz and digitized using RHS2116 amplifier chips and an RHS2000 stimulation/recording controller (Intan Technologies, USA). Data was then stored for offline analysis using Spike-2 software from CED.
Using electrode structure and electrode position within the cord, we estimated recordings to be superficial (laminae I–III) when they were obtained at a distance ≤ 260 μm from the dorsal border of the cord or deep (laminae IV and V) otherwise.
Signals from the MEA were amplified, band pass filtered between 200 and 3 KHz and digitized using RHS2116 amplifier chips and an RHS2000 stimulation/recording controller (Intan Technologies, USA). Data was then stored for offline analysis using Spike-2 software from CED.
Using electrode structure and electrode position within the cord, we estimated recordings to be superficial (laminae I–III) when they were obtained at a distance ≤ 260 μm from the dorsal border of the cord or deep (laminae IV and V) otherwise.
4
Single-cell RNA-Seq of Scleraxis-Positive Cells
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Control, non-injured Tnmd−/−ScxGFP+ and WT ScxGFP+ Achilles tendons (n = 2) were explanted and GFP+ cells were isolated by collagenase digestion (8 h) and filtration according to [22 (link)]. Next, cells (n = 20/genotype) were resuspended in PBS, placed on Adcell diagnostic slides (Thermo Fisher, Waltham, Massachusetts, USA), picked up in 1 µl PBS each using a micromanipulator (Patchman NP2) with pump (CellTram, both Eppendorf, Hamburg, Germany) and subsequently stored in Smart-Seq2 lysis buffer at −80 °C. The whole transcriptome amplification (WTA) and Illumina Nextera XT library preparation (Illumina, San Diego, California, USA) were performed as described by Picelli et al. [32 (link)]. The libraries were quantified using the KAPA Library Quantification kit (Roche Diagnostics, Mannheim, Germany), pooled in equimolar amounts and sequenced paired-end with read length of 2 × 150 bp and yield of 30 million reads per library on an Illumina HiSeq. In total, six ScxGFP+ cells (n = 6/genotype) were subjected to scRNA-Seq. Bioinformatic analysis is described in Supplementary information.
5
Intracellular FCS Measurements in Cockroach Salivary Glands
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In order to perform intracellular FCS measurements, salivary gland tissue of the American cockroach Periplaneta americana was dissected in physiological saline. Subsequently, the tissue was attached to a glass cover slip using the tissue adhesive Vectabond (Axxora, Lörrach, Germany) and the recording chamber was then mounted on the microscope stage. For dye injection, micropipettes with tip sizes of approximately 2 μm were prepared from glass tubes with filaments (GB150F-10, Science Products, Hofheim, Germany) using a micropipette puller (P-97, Sutter Instruments, Novato, USA). Micropipettes were loaded with 100 nM streptavidin labeled with AlexaFluor647 and injection into salivary gland cells was performed with an injection system (FemtoJet, PatchMan NP2, Eppendorf, Hamburg, Germany) under microscopic view (IX71, Olympus, Hamburg, Germany).
6
Microinjection of M. lignano Embryos
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All microinjections were carried out on fresh one-cell stage M. lignano embryos. An AxioVert A1 inverted microscope (Carl Zeiss, Germany) equipped with a PatchMan NP2 for the holder and a TransferMan NK2 for the needle (Eppendorf, Germany) was used to perform all of the micromanipulations. A FemtoJet express (Eppendorf, Germany), with settings adjusted manually based on the amount of mucous and debris surrounding the embryos, was used as the pressure source for microinjections. A PiezoXpert (Eppendorf, Germany) was used to facilitate the penetration of the eggshell and the cell membrane of the embryo.
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