TITLE: QGP-1 cell line  maintenance protocol 
AUTHORS: bellampalli.shreya  

[DESCRIPTION]
QGP-1 cell line  maintenance protocol; thawing and passaging QGP-1 cells

[STEPS]
SECTION: Thawing protocol
1. Warm media
SECTION: Thawing protocol
2. Swirl cells in 37 degree water bath until vial is thawed
SECTION: Thawing protocol
3. Add 7 mL of culture media to a T25 flask and gently pipette thawed cells into flask
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4. Let cells adhere for 24 hours and replace half of the culture media with fresh media
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5. Replace the media in the flask with fresh media after 48 hours
SECTION: Thawing protocol
6. Split cells once cells reach 70-80% confluency: usually takes 3-4 days from thawing and plating in a T25 until they are ready for passage. (they won’t ever become completely confluent, they will just start to grow on top of each other)
SECTION: Thawing protocol
7. Lift cells using trypsin and plate all cells in a T75
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8. They are usually ready to passage 3 days later.
SECTION: Passaging cells
51. Passaging Protocol
SECTION: Passaging cells
51. Warm media and trypsin in water bath
SECTION: Passaging cells
51. Clean hood with EtOH
SECTION: Passaging cells
51. Remove media
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51. Wash with HBSS
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51. Discard HBSS, add Trypsin, and place flasks in incubator for 5 minutes
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51. Bring flasks back to hood, obtain lifted cells and spin at 500 rcf for 5 minutes
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51. Discard supernatant
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51. Resuspend in 10 mL
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51. Place 250 uL of cells into a T75 flask and add
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51. mL of media
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51. 10.   Place flask back in incubator and split next week      
SECTION: Passaging cells
51. Matrigel Coating for QGP1
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51. Warm media and trypsin in water bath
SECTION: Passaging cells
51. Clean hood with EtOH
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51. Thaw matrigel overnight by submerging aliquot in ice and placing in 4 °C Fridge.
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51. Using cooled pipettes and working on ice, add 5% w/v of Matrigel to serum free RPMI Medium
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51. Place the minimum amount of the matrigel mix to cover the culture dishes
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51. a.      T75: 3mL
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51. b.      T25: 1mL
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51. c.       Mattek dishes: 250uL in inner circle
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51. Place plates at 37 °C for 1 to 2 hrs
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51. While plates are incubating at 37 °C, begin lifting cells as described above
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51. Count cells and resuspend in desired amount of media
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51. Right before plating cells, remove the matrigel solutions from the bottom of the new dishes (take care not to dry out)
SECTION: Passaging cells
51. 10.   Place desired amount of cell suspension into the matrigel coated dish
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51. 11.   Let cells attach overnight before using or let grow in incubator for the next week and passage again.           Notes about cells:  The cells tend to grow pretty slow and in clumps, but adhere very well and will be tough to lift off of the flask. For the plating and then first passaging I try to be gentle with the cells and keep them at a high density. The initial thawing/plating should be in a T25 or your cells will end up dying and the first passage should only be from a T25 to a T75. After those two steps, they are usually growing pretty well and ready to use. After this point you should expand into multiple flasks to expand the cells for freezing or experiments. If you are looking to generate your own supply, then I try to freeze cells after the first or second passage and freeze anywhere from 2,500,000 to 5,000,000 per vial. For weekly maintaining, I usually take about 1:40 of the 10 mL cell suspension or a total of 1,000,000 cells in a T75, and then they get to passaging confluency a week later.  If you will need them sooner than one week, then you can adjust the density from 1:10 to 1:20.  We typically stop using them after passage 23.     General Tips for Better Results:
SECTION: Passaging cells
51. Coat on Matrigel
SECTION: Passaging cells
51. Keep cells single and away from other cells. Don’t let them overgrow.
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51. Starting using for experiments 2 passages after thawing and then they are good for about 7-10 passages after that.   
SECTION: passaging protocol
9. 
SECTION: passaging protocol
9.1. Warm media and trypsin in water bath
SECTION: passaging protocol
9.2. Clean hood with EtOH
SECTION: passaging protocol
9.3. Remove media
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9.4. Wash with HBSS 
SECTION: passaging protocol
9.5. Discard HBSS, add Trypsin, and place flasks in incubator for 5 minutes
SECTION: passaging protocol
9.6. Bring flasks back to hood, obtain lifted cells and spin at 500 rcf for 5 minutes
SECTION: passaging protocol
9.7. Discard supernatant
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9.8. Resuspend in 10 mL
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9.9. Place 250 uL of cells into a T75 flask and add 13 ml of media 
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9.10. Place flask back in incubator and split next week