Jul 15, 2024
Senescence induction by DNA-damage in PCLS
- 1University of Pittsburgh
Protocol Citation: Delphine Beaulieu, Nayra Cardenes, Melanie Königshoff 2024. Senescence induction by DNA-damage in PCLS. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwdwpwlmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 22, 2024
Last Modified: July 15, 2024
Protocol Integer ID: 95638
Keywords: PCLS, Senescence, SenNet, Bleomycin, Doxorubicin, TriState SenNet, Lung
Funders Acknowledgement:
TriState SenNET (Lung and Heart) Tissue Map and Atlas consortium - NIA
Grant ID: U54AG075931
Abstract
Precision-Cut Lung Slices (PCLS) are uniform, in size and thickness, tissue slices generated from human lungs that can be used for disease modeling, drug discovery and preclinical validation, while retaining the lung's cellular complexity and tissue architecture.
This protocol describes two different methods for the induction of cellular senescence by DNA damage in PCLS, and collection of samples for different analysis and measurements.
Image Attribution
CLAR (Center for Lung Aging and Regeneration) Logo designed by Nayra Cardenes, PhD.
Guidelines
All steps involving human tissue must be conducted under BSL2 safety guidelines.
Protocol materials
Penicillin-StreptomycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #P00781
Step 1
Amphotericin B solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #A2942
Step 1
WST-8 Assay Kit (Cell Proliferation)AbcamCatalog #ab65475
Step 5.2
Senescence β-Galactosidase Staining KitCell Signaling TechnologyCatalog #9860
Step 5.3
Dimethyl sulfoxide (DMSO)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D2438-50ML
Step 4.2
Doxorubicin hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #D1515-10MG
Step 4.2
DMEM/F-12, powder, HEPESThermo Fisher ScientificCatalog #12400024
Step 1
16% Formaldehyde (w/v), Methanol freeLife TechnologiesCatalog #28908
Step 5.1
BleomycinFresenius KabiCatalog #10361
Step 4.2
Safety warnings
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Before start
To start this procedure, you must have PCLS (Precision-Cut Lung Slices) ready (check protocol DOI dx.doi.org/10.17504/protocols.io.36wgq3xr5lk5/v1).
Day -1: PCLS acclimation to growth conditions
Day -1: PCLS acclimation to growth conditions
Add 1 mL of media per well in 24-well plates DMEM F-12 media (DMEM/F-12, powder, HEPESThermo Fisher ScientificCatalog #12400024 ) + 1% FBS + 1% Penicillin-Streptomycin Penicillin-StreptomycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #P00781 + 0.3 µg/mL Amphotericin B Amphotericin B solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #A2942 .
Place one PCLS (1 cm diameter / 300 µm thick) per well with a brush.
Incubate o/n (overnight) at 37 oC, 5% CO2.
Treatment at Days 0, 2 and 4
Treatment at Days 0, 2 and 4
Treatment: change media with 1ml/PCLS, diluted in DMEM media + 0.1% FBS + 1% Pen/Strep + 1% Amphotericin B
Prior to media exchange, collect supernatant (s/n) from each condition in 1.5 mL tubes and, store at -80oC for future analysis.
Change media for each condition:
Control - Change media (above).
Bleomycin - 15 μg/ml Bleomycin in media above.
BleomycinFresenius KabiCatalog #10361
DMSO - 1:100,000 DMSO in media above.
Dimethyl sulfoxide (DMSO)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D2438-50ML
Doxorubicin - 0.1 μM Doxorubicin in media above.
Doxorubicin hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #D1515-10MG
PCLS Harvesting for measurements
PCLS Harvesting for measurements
Collect PCLS at Day 0, 2, 4 and Day 6 for different baseline and final timepoint measurements.
Days 0, 2,4 6:
Fix PCLS in 4% formaldehyde/PBS for 30 minutes (2x PCLS per condition).
16% Formaldehyde (w/v), Methanol freeLife TechnologiesCatalog #28908
Then wash twice with PBS and store at 4oC in PBS.
Use 1-2 slices for paraffin embedding, and posterior histological analysis.
Days 0 and 6:
Cell proliferation measurement: Use 2-3x PCLS to make 4x4 mm punches to perform WST-8 cell proliferation assay in a 96-well plate, following manufacturer's instructions.WST-8 Assay Kit (Cell Proliferation)AbcamCatalog #ab65475
Days 0 and 6:
Fix 2x PCLS with 1x fixative solution from β-galactosidase kit and stain for β-galactosidase as per manufacturer instructions.Senescence β-Galactosidase Staining KitCell Signaling TechnologyCatalog #9860
Day 0 and 6:
Snap freeze in liquid nitrogen: 4x PCLS per cryotube.
Store at -80oC for future analysis.