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Agarose-Based Preparation of Human Precision-Cut Lung Slices
In This Article
Overview
In this video, we describe a procedure to inflate isolated human lungs with agarose and prepare human precision-cut lung slices. The prepared ex vivo lung slices can be used for further immunologic experiments.
Protocol
All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.
1. General Preparation of Human PCLS and Subsequent Exposure to Chemicals
NOTE: Two persons are required to fill a lung. An up-to-date vaccination record for hepatitis A and B is recommended. Patients are routinely screened for HCV and HIV prior to lung transplantation. If an active infection with Mycobacterium tuberculosis is diagnosed or suspected, the lung should be rejected. Nevertheless, all fresh human lung tissue and samples derived from it must be t....
Disclosures
Materials
| Name | Company | Catalog Number | Comments |
| Silicon hose 3.0 x 5.0 mm | A. Hartenstein (Leipzig, Germany) | SS04 | |
| Syringe | Faust Lab Science (Klettgau, Germany) | 9.410 050 | |
| Coring tools | custom-made | Custom-made | |
| Trimming Blade Handle (FEATHER) | pfm medical ag (Cologne, Germany) | 205530001 | |
| Trimming Blades (FEATHER) | pfm medical ag (Cologne, Germany) | 205500000 | |
| Microtome: Tissue Slicer | Alabama R&D (Bad Homburg, Germany) | 303400-ADPT | Krumdieck Tissue Slicer (MD6000) |
| Microtome blade | Wilkinson Sword (Solingen, Germany) | ENR-4027800011506 | |
| Tissue culture dishes | Sigma (München, Germany) | Z666246-420EA | |
| Inoculation loop | Copan Diagnostics (Murrieta, USA) | CD176S01 | |
| TPP Tissue culture plates 24wells | Sigma (München, Germany) | Z707791-126EA | |
| Cassette | Thermo Scientific (Schwerte, Germany) | 1000957 | |
| Nunc MaxiSorp flat-bottom | Fisher Scientific GmbH (Hannover, Germany) | 44-2404-21 | |
| Nunc MicroWell 96-Well | Thermo Scientific (Schwerte, Germany) | 260836 | |
| Reagents | |||
| Agarose, low gelling temperature | Sigma (München, Germany) | A9414-100G | |
| Balanced Salt Mixtures and Solutions, Cell Culture, Classic Media and Salts, Earle's Balanced Salts (EBSS) | Sigma (München, Germany) | E2888-500ML | |
| Penicillin and streptomycin | Lonza (Verviers, Belgium) | 17-602E | |
| Dulbecco´s Modified Eagle´s Medium Nutrient Mixture F-12 Ham (DMEM F-12) | Gibco (Darmstadt, Germany) | 11039-047 | Culture medium |
| Dulbecco´s Phosphate with Ca and Mg (DPBS) | Lonza (Verviers, Belgium) | BE17-513F |
This article has been published
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Source: Neuhaus, V. et al., Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices. J. Vis. Exp. (2018)
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