Hi, I am going to start with RAW264.7 cultures. I would like to know if I can detach them with trypsin/EDTA?? and if it is better to culture them in flask or in petri dishes?? thanks so much!
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#2
rhombus
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Posted 21 September 2010 - 01:50 AM
Dear Alvaro,
RAW's stick to TC treated plastic like super glue and are very difficult to get off with Trypsin/Versene. We grow them in suspension very sucessfully in Techne Biological stirrers. If you cannot get hold of a stirrer I would suggest using NON TC treated plastic.
I hope this is useful.
Kindest regards,
Uncle Rhombus
RAW's stick to TC treated plastic like super glue and are very difficult to get off with Trypsin/Versene. We grow them in suspension very sucessfully in Techne Biological stirrers. If you cannot get hold of a stirrer I would suggest using NON TC treated plastic.
I hope this is useful.
Kindest regards,
Uncle Rhombus
#3
alvaro
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Posted 27 September 2010 - 03:08 AM
Ok. I only have TC-treated Flask now, so I supose they will attach strongly. Are there other options like scrapping?? or you dont recomend scrapping this type of cells?? If it is possible to culture RAW264.7 cells with TC-treated Flask and scrapping them...could you pass me an appropriate protocol for scrapping the cells??
thanks so much!
thanks so much!
#4
rhombus
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Posted 28 September 2010 - 02:00 AM
alvaro, on 27 September 2010 - 03:08 AM, said:
Ok. I only have TC-treated Flask now, so I supose they will attach strongly. Are there other options like scrapping?? or you dont recomend scrapping this type of cells?? If it is possible to culture RAW264.7 cells with TC-treated Flask and scrapping them...could you pass me an appropriate protocol for scrapping the cells??
thanks so much!
thanks so much!
RAW cells should be grown in suspension. If you scrape them you will reduce the total viability from 99% (suspension) down to 50% (scraped). The dead cells will also release intracellular contents that might affect the viable cells? You will also be putting a "selection" pressure on the cells i.e. cells that are more resistant to scraping will be selected.
Kindest regards.
Uncle Rhombus
#5
alvaro
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Posted 29 September 2010 - 01:19 AM
Ok. I will try to culture them in suspension...thanks for your help!
#6
Clare
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Posted 29 September 2010 - 01:34 AM
alvaro, on 29 September 2010 - 01:19 AM, said:
Ok. I will try to culture them in suspension...thanks for your help!
Our lab cultures them on square Sterilin dishes and just 'blows' the cells off the surface: by filling a syringe (and wide needle) with media and washing the surface (whilst the plate is on an angle).
Clare
