A Standardized Protocol for Inducing Stress-Induced Premature Senescence in Primary Human Melanocytes Using Tert-butyl Hydroperoxide

Our research focuses on investigating the senescence phenotype of melanocytes and its contribution to extrinsic skin aging. Established senescence models use fibroblasts and failure to capture melanocyte-specific biology. This protocol induces tBHP-mediated oxidative stress in primary human melanocytes.

To begin, gather the required reagents. After thawing the melanocytes and allowing them to recover for three days, ensure that the cells reach 80 to 90%confluency on the seeding day. Aspirate the medium from the flask.

Wash the cells twice with DPBS. Add two milliliters of trypsin EDTA solution to detach the cells. Place the flask in the incubator for five minutes at 37 degrees Celsius with 5%carbon dioxide.

Gently tap the flask. And under the microscope, observe the cells to confirm complete detachment. Add 1.5 times the volume of trypsin inhibitor to stop the reaction.

Transfer the cell suspension to a 15-milliliter conical tube. Dilute the suspension with DPBS to a final volume of 10 milliliters. Centrifuge the cell suspension at a relative centrifugal force of 100 for five minutes.

Aspirate the supernatant and re-suspend the cell pellet in medium to the desired volume. Seed 600, 000 cells per 10 centimeter dish for tert-Butyl hydroperoxide, or tBHP, treated plates. Seed 400, 000 cells per plate for the control plates.

Prepare a 10 millimolar working solution from the tBHP stock using DPBS or media. Add 100 micromolar tBHP to the cells for one hour per treatment. Move the plate back and forth to distribute the tBHP evenly throughout the medium.

Place the cells in the incubator for precisely one hour at 37 degrees Celsius with 5%carbon dioxide. Aspirate the medium to terminate the tBHP treatment. Wash the plates twice with preheated DPBS at 37 degrees Celsius.

Add the appropriate volume of fresh medium to the plates. Place the plates in the incubator and allow the cells to recover for at least four hours before the next treatment. Repeat the treatment procedure later on the same day for the second treatment.

Continue the treatment regimen through day four, then maintain the cells in culture until day nine to assess senescence markers. Count the tBHP-treated cells and control cells. Reseed them at the same initial density, or if fewer than 600, 000 cells are obtained from the tBHP-treated plates, reseed all collected cells.

Count the cells on days four, nine, and 15 of culture. On day nine, wash the cells twice with PBS. Fix the cells using 2%formaldehyde and 0.4%glutaraldehyde.

Wash the fixed cells twice with PBS. Incubate the fixed cells in a staining solution at pH 6. Cover the plate to protect it from light, then incubate them for 18 hours at 37 degrees Celsius in the absence of carbon dioxide.

Wash the cells twice with PBS. Using light microscopy, image and count the number of blue stained cells. Divide it by the total number of cells within the same field.

Express the results as the percentage of positive cells. Following oxidative stress induction, tBHP treatment significantly reduced melanocyte proliferation compared to controls. The treated cells reached only 1.4 cumulative population doublings, or cPDL, by day nine, whereas controls reached 3.1 cPDL.

By day 15, treated cells plateaued at 1.9 cPDL, while control cells expanded to 5.1 cPDL. Melanocytes treated with tBHP showed increased cell surface area compared to controls at all measured time points, with enlargement progressing until day 15, consistent with the enlarged and flattened morphology of senescent cells. tBHP-treated melanocytes showed an increase in senescence-associated beta-galactosidase, or SA-beta-galactosidase activity, with 32%positive cells compared with 3%in controls.

The rise in SA-beta-galactosidase positivity was consistent with a senescent phenotype. Immunofluorescence analysis showed increased gamma-H2AX levels in tBHP-treated melanocytes compared with controls. The treated cells showed stronger gamma-H2AX signal than controls.

Real-time quantitative polymerase chain reaction analysis demonstrated increased senescence-associated secretory phenotype factor expression, with interleukin-8 showing the largest increase. While interleukin-6 and matrix metalloproteinase-1 showed moderate increases. Western blot analysis confirmed senescence induction.

Lamin B1 showed a significant reduction in tBHP-treated melanocytes compared to controls. Phosphorylated retinoblastoma protein levels were reduced in tBHP-treated melanocytes compared to controls. tBHP treatment increased transcriptional expression of p16 and p21 in melanocytes.

This model allows researchers to study how senescence melanocytes affect pigmentation and communicate with surrounding cells via SASP inflammatory factors. The challenge in this protocol is precise timing. tBHP must be applied twice daily with at least four hours interval.

Following this protocol, senescence can be characterized using senescence-associated beta-galactosidase staining, immunofluorescence, RT-qPCR, and Western blotting of established senescence markers.