TITLE: LD-HD Pigmentation Oscillator Model
AUTHORS: Vivek T Natarajan

[DESCRIPTION]
LD-HD pigmentation oscillator model uses B16 mouse melanoma cells which are seeded at a very low seeding density of 100 cells/cm2 and 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.

[GUIDELINES]
Cell Counting Guideline:

Number of cells in 1 quadrant= Number of cells present in 10-4ml/0.1µl of media
(10-4 ml is actually the volume of hemocytometer quadrant.
{Each square width =250µm. so, for 4 squares in a quadrant}

i.e. 4 * 250µm =1000µm or 1mm
Length of each quadrant= 1mm
Width of each quadrant= 1mm
Height of hemocytometer chamber= 0.1mm
Volume of chamber = 0.1 mm3 =10-4 ml
10-4 ml = 0.1µl

Number of cells in 1ul is therefore = 10 X Number of cells counted in one quadrant
Number of cells in 1ml is therefore = 104 X Number of cells counted in one quadrant

[STEPS]
SECTION: FOR LD (Low Density) CYCLE OF PIGMENTATION
1. 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.
SECTION: FOR LD (Low Density) CYCLE OF PIGMENTATION
2. Discard media using a steri-pipette.
SECTION: FOR LD (Low Density) CYCLE OF PIGMENTATION
3. Wash the cells with 3-5ml of dPBS
SECTION: FOR LD (Low Density) CYCLE OF PIGMENTATION
4. Add 0.5-1ml of 0.1%trypsin solution to cover the entire area of flask base
SECTION: FOR LD (Low Density) CYCLE OF PIGMENTATION
5. Incubated for 2 minutes at 37ᵒC for cells to trypsinize
SECTION: FOR LD (Low Density) CYCLE OF PIGMENTATION
6. Meanwhile, prepare DMEM+10%FBS media (45ml DMEM +5ml FBS)
SECTION: FOR LD (Low Density) CYCLE OF PIGMENTATION
7. Take out the flask from incubator. Add 1ml of DMEM+ FBS (10%) to quench trypsin (Basically, double the volume of trypsin used).
SECTION: FOR LD (Low Density) CYCLE OF PIGMENTATION
8. Collect the suspension in a 15 ml falcon.
SECTION: FOR LD (Low Density) CYCLE OF PIGMENTATION
9. Centrifuge at 1000rpm for 7 minutes. Discard the supernatant and resuspend the pellet in few ml media.
SECTION: FOR LD (Low Density) CYCLE OF PIGMENTATION
10. Count the number of cells using hemocytometer.
SECTION: FOR LD (Low Density) CYCLE OF PIGMENTATION
11. Dilute the suspension if required. If the number of cells is less enough to be easily counted, no dilution is required.
SECTION: FOR LD (Low Density) CYCLE OF PIGMENTATION
12. 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.
SECTION: FOR LD (Low Density) CYCLE OF PIGMENTATION
13. Take the required number of flasks and label them properly adding date and name of user, cell type, passage number.
SECTION: FOR LD (Low Density) CYCLE OF PIGMENTATION
14. Add 5ml-10 ml of DMEM (10% FBS) media containing cells (master mix cell suspension prepared above) into each T25 or T75 respectively.
SECTION: FOR LD (Low Density) CYCLE OF PIGMENTATION
15. Incubate the flasks at 37ᵒC incubator under 5% CO2.
SECTION: FOR HD (High Density) CYCLE OF DEPIGMENTATION
16. Trypsinize the flasks on Day 12 of LD.
SECTION: FOR HD (High Density) CYCLE OF DEPIGMENTATION
17. 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.
SECTION: FOR HD (High Density) CYCLE OF DEPIGMENTATION
18. Change media on day 14 (if required).
SECTION: FOR HD (High Density) CYCLE OF DEPIGMENTATION
19. Collect the pellet on Day 16 and Day 20 of LD.
Gradual depigmentation of cell pellet will be observed.