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Daniel:Notebook/HiResChrPaint/2013-9-24
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=Freezing Fibroblast Cells= [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] ===Protocol=== from [[Daniel:Protocols/FreezeCells|cell freezing protocol]] 1. Check cells for confluency 2. Aspirate, wash with PBS 3. Add 500 uL trypsin, incubate 3 minutes 4. Add 7.5 of media and pool as you go (for five dishes you’ll end up with 10 mL) 5. Take 200 uL and plate it (in 10 mL media); add 10 uL cell suspension and 90 uL DMEM to petri dish 6. Centrifuge 3 minutes at 1000 rpm 7. Aspirate media **Do the next steps fast! 8. Prepare a fresh tube with 6 mL of 5%DMSO in DMEM; try to find DMSO in Alan’s box or next to tryp LE 9. Resuspend the cells in 5 mL and aliquot 1 mL into each vial 10. Initials, Date, Cell Line, Passage; top and side 11. Put into Mr. Frosty; Transfer to -80 12. Next day transfer to liquid nitrogen tank for long-term storage (ask Chris about a box, we use rack 1) ==Fibroblast Adherence== After 5 hours I looked at the cells in the petri dishes. They seemed to be growing well. So I decided to try fixing a few. ===Protocol=== [[Daniel:Protocols/FISH#Slide Preparation|FISH Slide Preparation Protocol]] 0. Replaced media on dishes 3 and 4 (aspirate media and add 1 mL DMEM) 1. Rinse slides briefly in 1X PBS 2. Fix slides for 5-15 minutes in 4% (v/v) paraformaldehyde in 1X PBS 3. Rinse briefly in 1X PBS in a coplin jar 4. Incubate for 5 minutes in 2X SSCT 5. Incubate for 5 minutes in 2X SSCT + 50% (v/v) formamide 6. Transfer to fresh container of 2X SSCT + 50% (v/v) formamide for storage at 4C 7. Slides are typically best used within 1-2 weeks of creation
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