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PixCell IIe

Manufactured by Arcturus
Sourced in United States

The PixCell IIe is a laser-based microdissection system designed for precise and efficient cell isolation. It features a high-precision laser capture mechanism that allows for the selective extraction of targeted cells or tissue samples from heterogeneous samples. The PixCell IIe is a laboratory instrument used for micromanipulation and microscale sample preparation.

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6 protocols using PixCell IIe

1

Laser Capture Microdissection of Osteoarthritic Cartilage

For LCM, cartilage tissues were obtained from 16 OA knees and 9 control knees, at 3–5 sites per knee. OA cartilage samples were harvested from both the macroscopically intact areas (preserved areas) and the areas showing various degrees of cartilage degeneration (degenerated areas). At each site, approximately 20 mm × 5 mm of cartilage was obtained in full thickness above the tide mark. The separation of cartilage zones by LCM was performed following a previously described method [5 (link), 24 (link)]. In brief, cryosections were prepared from the cartilage tissues in a plane vertical to the joint surface, which were separated into three cartilage zones using an LCM device (PixCell IIe; Arcturus, Mountain View, CA, USA) based on the histological features.
Immediately after LCM, RNA was extracted from the respective cartilage zones using an RNeasy Micro kit (Qiagen GmbH, Hiden, Germany) with routine use of DNase I (Qiagen). cDNA was synthesized using Sensiscript reverse transcriptase (Qiagen). The gene expression was evaluated quantitatively by real-time PCR on a LightCycler (Roche Diagnostics, Basel, Switzerland), using gene-specific primers and probes (Additional file 1: Table S1). SYBR® Premix Ex Taq® Perfect Real Time (Takara Bio, Shiga, Japan) or Premix Ex Taq® Perfect Real Time (Takara Bio) was used for PCR. The cDNA levels were normalized by the expression of GAPDH.
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2

Laser Capture of IPMN Cells for Mitochondrial DNA Analysis

FFPE specimens of IPMN slides (n=4) were subjected to laser capture micro-dissection. After evaluation for Ki-67 positivity, cells were captured using Arcturus PixCell IIe (Arcturus, CA, USA). In each patient, 3–4 lesions were selected. Ten to 30 cells were captured and their DNA extracted using QIAamp DNA FFPE Tissue Kit (Qiagen). The extracted DNA was amplified using a Single Cell Whole Genome Amplification Kit (WGA4; Sigma). DNA was further amplified by PCR reaction using 6 overlapping primer pairs for the D-loop of the mitochondrial genome (Supplemental Table 2). PCR products were cleaned by ExoSAP-IT (Affymetrix), followed by bidirectional Sanger sequencing on an ABI 3730XL DNA Analyzer.
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3

Isolation and Analysis of B Cell Lineage from CNS Tissue

Central nervous system tissue was sectioned at 12 μm on a microtome/cryostat, mounted onto a glass slide then fixed in 75% ethanol for 30 s. For the identification and capture of individual B lineage cells, the tissue was stained with mouse anti-CD20 or anti-CD38 antibodies (Accurate Chemical & Scientific) after fixation, then counterstained with poly-horseradish peroxidase anti-mouse IgG (Ivax Diagnostics). The tissue was then dehydrated in consecutive washes of 75, 95, and 100% ethanol then xylene. Cells were captured with a PixCell IIe laser capture microdissection instrument and CapSure Macro caps (Arcturus) and immediately stored at −80°C. RNA was isolated with the Absolutely RNA Nanoprep Kit (Stratagene) according to the manufacturer’s protocol. B cell variable regions were cloned and analyzed according to procedures that we have previously reported (18 (link), 33 (link)).
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4

Transcriptomic Analysis of Chlorine-Exposed Mouse Trachea

Frozen sections were prepared from distal trachea collected from five mice 4 days after chlorine exposure and from five unexposed mice. Laser capture microdissection with an Arcturus PixCell IIe instrument was used to isolate subepithelial tissue from hematoxylin and eosin-stained frozen sections from chlorine-exposed and unexposed mice based on published methods (Andres and Wittliff, 2011 (link)). Tissue was dissected from 10 sections per mouse. RNA was prepared from dissected tissue using an Arcturus® PicoPure® extraction kit (ThermoFisher Scientific, Waltham, MA) and analyzed on an Agilent 2100 Bioanalyzer (Santa Clara, CA). For transcript expression analysis by microarray, 250 ng of total RNA from each sample was amplified and labeled according to the GeneChip® WT PLUS Reagent kit protocol from Affymetrix (ThermoFisher, Waltham, MA), followed by hybridization to Affymetrix Mouse Gene 2.0 ST® arrays. The arrays were processed following the manufacturer recommended wash and stain protocol on an Affymetrix FS-450 fluidics station and scanned on an Affymetrix GeneChip® 7G scanner using Command Console 4.0. Gene expression data from the microarray analysis were submitted to the NCBI Gene Expression Omnibus (GEO) repository with accession number GSE109365.
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5

Laser Capture Microdissection and Whole Genome Amplification

Sections of PFC were cut at 8 μm thick onto Arcturus HistoGene Slides at −20°C for LCM on a Leica CM 1950 Cryostat after being embedded in M1 Embedding Matrix (Thermo Scientific). Staining of the slides was done with the Arcturus HistoGene Frozen Section Staining Kit (Life Technologies) using the manufacturer protocol. Laser Capture Microdissection was performed on an Arcturus PixCell IIe with CapSure HS LCM caps. Capturing was done at 20× optics using a 15 μm spot size. The target parameter was set to 0.200 V with a power of 35 mW and a duration of 0.7 ms. Ten cells of a specific type were captured per cap followed by lysis directly on the cap. Whole genome amplification was performed using a user-developed protocol of the Repli-g Mini Kit (Qiagen) with a 16 hour amplification time. DNA clean up was done using the QIAmp DNA Micro Kit (Qiagen) and quantified using Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies). Deletion validation PCR was done using 100 ng template material.
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Laser Capture Microdissection was done with an Arcturus PixCell IIe. Target parameters were set to 0.200 V with a 0.7 ms duration at 35 mW power. Images were captured using the LCM's built in CCD camera (Hitachi K.P-D590-V1) and processed using Arcturus' LCM control software (version 2.0). DNA agarose gel pictures were taken using an 8-megapixel digital camera. Color images were then converted to greyscale using Adobe Photoshop software.
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