Jul 28, 2025
Isolation of pure neuromelanin extracts from cultured TR5TY6 neuroblastoma cells
- Iria Carballo-Carbajal1,
- Marta Gonzalez-Sepulveda1,
- Joana M. Cladera-Sastre1,
- Miquel Vila2
- 1Vall d'Hebron Research Institute;
- 2VHIR-CIBERNED-ASAP
- Vilalab Public
Protocol Citation: Iria Carballo-Carbajal, Marta Gonzalez-Sepulveda, Joana M. Cladera-Sastre, Miquel Vila 2025. Isolation of pure neuromelanin extracts from cultured TR5TY6 neuroblastoma cells. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vz93p5gx1/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: July 28, 2025
Last Modified: July 28, 2025
Protocol Integer ID: 223402
Keywords: isolation of pure neuromelanin extract, pure neuromelanin extract, neuromelanin, cultured tr5ty6 neuroblastoma cells isolation
Funders Acknowledgements:
ASAP
Grant ID: ASAP-020505
Abstract
Isolation of pure neuromelanin extracts from cultured TR5TY6 neuroblastoma cells
Troubleshooting
Cell culture
Seed TR5TY6 cells in 12 cm diameter plates using 20 ml of medium per plate. 10 big plates will yield approximately 1-2 mg of pigment, depending on confluence.
Next day, induce tyrosinase expression by changing the medium for a new one containing 2 ug/ml doxycycline (stock 2 mg/ml).
Incubate the cells for 6 days. DO NOT CHANGE MEDIUM during this time, since neuromelanin accumulates not only in living cells, but also in dying cells and media.
Harvest the cells by using a cell scraper and collect them together with the medium in 50 ml tubes. Wash all the plates with extra medium (10 ml) and add it to the tubes.
Centrifuge at 1800 rpm for 7 min and discard supernatant.
Wash pellets with 10 ml PBS
Centrifuge again at 1800 rpm for 7 min, discard the supernatant and freeze immediately at -80ºC.
Neuromelanin isolation
Thaw cell pellet at RT
Wash in 10 ml of 0.05 N phosphate buffer pH 7.2 (2-3 pellets)
Centrifuge at 10,000 x g for 15 min at RT
Repeat washing and centrifugation steps
Add 10 ml of 5 mg/ml SDS (freshly added during the previous centrifuge) in 75 mM Tris, pH 7.5
Sonicate (until total resuspension) and incubate for 3 h at 37°C in a shaking water bath.
Centrifuge at 10,000 x g at RT for 30 min and remove the supernatant.
Add 2 ml (2-3 pellets) of 5 mg/ml SDS in 75 mM Tris, pH 7.5 with 0.33 mg/ml proteinase K (82.5ul in 5ml) and incubate for 3 h at 37°C
Transfer the samples to Eppendorf tubes, centrifuge at 10,000 x g at RT for 30 min and remove the supernatant.
Wash pellets with 1 ml 0.9% NaCl
Centrifuge at 10,000 x g for 30 min at RT and discard the supernatant.
Resuspend each pellet in 1 ml of MQ H2O (use the same ml for every pellet, taking it form one tube to the next one), pool them together in one tube and separate 25 ul of the resuspended sample for nanodrop quantification. Store it at 4ºC.
Centrifuge at 10,000 x g at RT for 30 min and remove the supernatant.
Rewash pellets in 1 ml of methanol. DO NOT resuspend the pellet.
Centrifuge at 10,000 x g at RT for 30 min and discard the supernatant.
Wash with 1 ml of hexane DO NOT resuspend the pellet.
Centrifuge at 10,000 x g at RT for 30 minutes.
Dry isolated NM under fume hood (be careful not to overdry the sample), fast-freeze by liquid nitrogen immersion and store at -80°C.