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=Fibroblast Adherence ([[Daniel:Notebook/HiResChrPaint/2013-9-9|Started 09/09/2013]])= [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] ==Testing Cell Seeding Density== I think the only parameter I want to test now is cell seeding density. Typical protocol I let the cells grow to confluence in petri dishes then trypsinize in 500 uL, followed by resuspension in 9.5 mL DMEM. This yields approximate cell density of 1E6 cells/mL. The paper (Beliveau et al) recommends cell seeding density of 5E5-2E6 cells/mL. I'm going to explore the cell seeding densities, therefore, at 1/2 density as usual (500,000 cells/mL) and 2x density (2 million cells/mL). I'm also going to test the kicker theory. I'll take timecourse for the first 1-3 hours, since that is what the original protocol called for. If the experiment does not go well today, I'll repeat tomorrow early morning and do a longer timecourse ===Protocol=== 1. Aspirate media and rinse with PBS 2. Add 500 uL trypsin and incubate 5 min. 3. Resuspend cells in 9.5 mL media 4. Add 100 uL cells to dishes 1 and 4 5. Add 50 uL cell suspension and 50 uL DMEM to dish 2 6. Spin down cells and resuspend in 250 uL trypsin + 4.75 mL DMEM; add 100 uL to dish 3 7. Timecourse at 1,2, and 3 hours post initial incubation 8. Add 100 uL DMEM to dish 4 1 hour post initial incubation ===Results===
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