Senescence induction by DNA-damage in PCLS

Melanie Königshoff, Nayra Cardenes, Delphine Beaulieu

Published: 2024-07-16 DOI: 10.17504/protocols.io.e6nvwdwpwlmk/v1

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.

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).

Steps

Day -1: PCLS acclimation to growth conditions

1.

Add 1 mL of media per well in 24-well plates DMEM F-12 media () + 1% FBS + 1% Penicillin-Streptomycin + 0.3 µg/mL Amphotericin B .

2.

Place one PCLS (1 cm diameter / 300 µm thick) per well with a brush.

3.

Incubate o/n (overnight) at 37 ^oC, 5% CO₂.

Treatment at Days 0, 2 and 4

4.

Treatment: change media with 1ml/PCLS, diluted in DMEM media + 0.1% FBS + 1% Pen/Strep + 1% Amphotericin B

4.1.

Prior to media exchange, collect supernatant (s/n) from each condition in 1.5 mL tubes and, store at -80^oC for future analysis.

4.2.

Change media for each condition:

Control - Change media (above).

Bleomycin - 15 μg/ml Bleomycin in media above.

DMSO - 1:100,000 DMSO in media above.

Doxorubicin - 0.1 μM Doxorubicin in media above.

PCLS Harvesting for measurements

5.

Collect PCLS at Day 0, 2, 4 and Day 6 for different baseline and final timepoint measurements.

5.1.

Days 0, 2,4 6:

Fix PCLS in 4% formaldehyde/PBS for 30 minutes (2x PCLS per condition).

Then wash twice with PBS and store at 4^oC in PBS.

Use 1-2 slices for paraffin embedding, and posterior histological analysis.

5.2.

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.

5.3.

Days 0 and 6:

Fix 2x PCLS with 1x fixative solution from β-galactosidase kit and stain for β-galactosidase as per manufacturer instructions.

5.4.

Day 0 and 6:

Snap freeze in liquid nitrogen: 4x PCLS per cryotube.

Store at -80^oC for future analysis.