UVB-induced senescence of HaCaT cells

Title: UVB‑induced senescence of HaCaT cells

Authors:
Jiayu Liu (Clarity0103@163.com)

Xiaobo Wang(wxb4320062@163.com)

School of Basic Medicine, Dali University, Dali, 671000, Yunnan, China

1. Abstract
This protocol describes an in vitro model of cellular senescence induced by ultraviolet B (UVB) irradiation in human immortalized keratinocytes (HaCaT cells). By applying two consecutive daily exposures of different UVB doses, the method mimics  photo‑damage and is suitable for studying skin photoaging and the effects of pharmacological interventions on senescence‑related signalling pathways.

2. Materials and Reagents

3. Equipment

4. Procedure

4.1 Pre‑irradiation warm‑up (critical step)

• Before each irradiation session, turn on the UV crosslinker and run it empty (without samples) for 5 minutes to stabilise the lamp output.

• After warming up, open the door and allow the chamber to ventilate briefly, then the instrument is ready for sample irradiation.

4.2 Cell seeding (Day –2)

1. Culture HaCaT cells in a 6‑cm dish until they reach >90% confluence.

2. Remove old medium, wash twice with PBS, trypsinise, and neutralise with complete medium (MEM + 10% FBS). Centrifuge at 1000 rpm for 5 min.

3. Discard supernatant and resuspend the cell pellet in 3 mL of complete medium to obtain a single‑cell suspension.

4. For a 24‑well plate: add 500 µL of complete medium to each well, then add 100 µL of cell suspension (final volume 600 µL/well). Gently swirl to distribute cells evenly.

5. Incubate at 37°C, 5% CO₂ for 24 hours.

4.3 First UVB irradiation (Day –1, dose 5 mJ/cm²)

1. Check cell density: after 24 h, cells should reach ~50–60% confluence.

2. Replace with PBS overlay: aspirate old medium from each well and add 200 µL of sterile PBS (ensure complete coverage of the cell layer).

3. Irradiate: remove the plate lid and place the plate into the pre‑warmed UV crosslinker.

   • Set exposure time: based on the instrument irradiance (2.5 mJ/cm²/s), use 2 seconds (total dose = 2.5 × 2 = 5 mJ/cm²).

4. Post‑irradiation medium change: immediately after irradiation, aspirate PBS and add 500 µL of fresh complete medium (with or without drug treatment, according to your experimental design).

5. Continue culture: return the plate to the incubator for another 24 hours.

4.4 Second UVB irradiation (Day 0, dose 2.5 mJ/cm²)

1. Check cell density: after the second 24‑h incubation, cells should be ~90–100% confluent (fully confluent).

2. Replace with PBS overlay: aspirate old medium and again add 200 µL of sterile PBS per well.

3. Irradiate: remove lid and irradiate for 1 second (total dose = 2.5 × 1 = 2.5 mJ/cm²).

4. Post‑irradiation medium change: immediately aspirate PBS and add 500 µL of fresh complete medium (with desired treatments).

5. Senescence model established: this time point is defined as Day 0. The cells are now ready for downstream assays such as SA‑β‑Gal staining, Western blotting, etc.

5. Important Notes and Limitations

⚠️ Crucial considerations for cross‑laboratory reproducibility:

1. Cell growth rate variability – Differences in serum lots, medium brands, or passaging habits can affect doubling time. Always perform a growth curve pilot to adjust seeding density so that the desired confluence (50–60% on Day –1, 100% on Day 0) is achieved in your own hands.

2. UV crosslinker model and pre‑experimental calibration (major risk) – This protocol uses the Scientz SCIENTZ03‑II with an output of 2.5 mJ/cm²/s. However, different UV crosslinker models can have substantially different irradiance outputs, and even the same model may show unit‑to‑unit variation. Therefore, we strongly recommend that researchers measure the actual irradiance of their own instrument using a UV radiometer to obtain the true energy output.
   More importantly, because cell growth conditions, culture vessel types, and other variables also influence the final effect, a pilot dose‑response experiment is essential to identify the optimal exposure parameters (both duration and total energy) for your specific system. Do not directly copy the “2‑second / 1‑second” times from this protocol without performing your own calibration and pilot tests. Always adjust the conditions based on your own measurements and validation experiments.

3. Culture vessel effects – Different plate formats (e.g., 6‑well vs. 24‑well) or dish sizes have different exposed surface areas and may alter UV transmission through the liquid overlay. When changing vessel types, perform a dose‑response pilot experiment to optimise the irradiation duration.

4. Protocol source and optimisation – This protocol is optimised based on the method described in "Vorinostat attenuates UVB‑induced skin senescence by modulating NF‑κB and mTOR signaling pathways". It differs from the original procedure in certain details (e.g., cell seeding density, irradiation doses, and schedule). Through long‑term practice in our laboratory, the current version has proven to be stable, reproducible, and reliable. Users are encouraged to adopt this optimised version as a reference.