LD-HD Pigmentation Oscillator Model
Vivek T Natarajan
pigmentation
B16 melanoma cells
depigmentation
progressive pigmentation model
pigmentation oscillator
Abstract
LD-HD pigmentation oscillator model uses B16 mouse melanoma cells which are seeded at a very low seeding density of 100 cells/cm2and are left without change in media. Cells under such conditions gradually starts pigmenting, beginning on Day 4 of LD. The cells survive up till day 12 of LD when they are trypsinised and reseeded at a higher density of 10000 cells/cm2. Owing to high density culture, these cells start losing their pigment and gradually become depigmented by Day 16-20 i.e. four to eight days after seeding at high density. This is a great model which allows segregation of different stages of pigmentation and subsequent molecular changes to be followed over a period of 20 days.
Steps
FOR LD (Low Density) CYCLE OF PIGMENTATION
Take a confluent B16 mouse melanoma cell culture maintained in DMEM+10%FBS media.
Confluency can be checked under a microscope and by change in color of media from pink to yellow-orange.
Discard media using a steri-pipette.
Wash the cells with 3-5ml of dPBS
Add 0.5-1ml of 0.1%trypsin solution to cover the entire area of flask base
Incubated for 2 minutes at 37ᵒC for cells to trypsinize
Meanwhile, prepare DMEM+10%FBS media (45ml DMEM +5ml FBS)
Take out the flask from incubator. Add 1ml of DMEM+ FBS (10%) to quench trypsin (Basically, double the volume of trypsin used).
Collect the suspension in a 15 ml falcon.
Centrifuge at 1000rpm for 7 minutes. Discard the supernatant and resuspend the pellet in few ml media.
Count the number of cells using hemocytometer.
Dilute the suspension if required. If the number of cells is less enough to be easily counted, no dilution is required.
For setting up an LD we require nearly 100 cells/cm2 i.e. 2500 per 4-5ml media in a T25 flask or 7500 cells in 10-12 ml media in a T75 flask.
Take the required number of flasks and label them properly adding date and name of user, cell type, passage number.
Add 5ml-10 ml of DMEM (10% FBS) media containing cells (master mix cell suspension prepared above) into each T25 or T75 respectively.
Incubate the flasks at 37ᵒC incubator under 5% CO2.
FOR HD (High Density) CYCLE OF DEPIGMENTATION
Trypsinize the flasks on Day 12 of LD.
Seed the cells at a higher density of 10000 cells/cm 2 i.e 800000 cells per T75.
Similarly, 250000 cells per T25 for HD.
Change media on day 14 (if required).
Collect the pellet on Day 16 and Day 20 of LD.
Gradual depigmentation of cell pellet will be observed.
