Hosuk:Cell fixation in culture dish: Difference between revisions

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#For cells in MatTek glass bottom dish(P/N : P35G-1.5-14-C)
*For cells in MatTek glass bottom dish(P/N : P35G-1.5-14-C)
#1mL of formalin (~39% formaldehyde)directly to 2 ml of culture dish, incubate for 15min at 37C
#NO aspirate media. add 200 ul formalin(~39% formaldehyde) to 2 ml media directly,  
#Change media of fresh formalin+PBS (1mL), incubate for 15min at 37C,
#incubate for 15 min at 37C
#Wash with PBS, and add 70% Ethanol (2mL)  
#aspirate
#Store at 4C
#quickly add pre warmed 10% formalin in PBS
#incubate at 37C for 15 min
#aspirate
#wash with PBS 3 times at RT
#incubate in 70% etoh for 1 min or store in 70% etoh up to a week at 4C
 
 
 
===Jonathan's protocol===
*100% confluence, and could wait one more day, and split
*When cells are transferred from 6 well plate
**500uL of Trypsin, incubate 5min
**Add 500uL media, and mix well, and
**100uL media in 96 well, or 300~350uL media in glass bottom dish
**Add 60uL of cell in 96 well or 200uL in glass bottom dish
**Culture one or two more day
 
 
*Fixed cells just using 10% Formamide
**Aspirate media
**Add 50uL of 10% Formamide (200uL for glass bottom)
**Incubate for 15min
**Wash with PBS three times
**Store in Ethanol
 
 
 
===PGP1F cell culture in 96 well plate===
*Add 1mL Trypsin in T25 flask (about 90~100% covered)
*Add 4mL media (total 5mL)
*Add 100uL cell (from 5mL in T25 flask) to each well in 96 well plate (total 16 wells)
*Add 200uL media to each well, and gently mix

Latest revision as of 17:15, 26 December 2013

  • For cells in MatTek glass bottom dish(P/N : P35G-1.5-14-C)
  1. NO aspirate media. add 200 ul formalin(~39% formaldehyde) to 2 ml media directly,
  2. incubate for 15 min at 37C
  3. aspirate
  4. quickly add pre warmed 10% formalin in PBS
  5. incubate at 37C for 15 min
  6. aspirate
  7. wash with PBS 3 times at RT
  8. incubate in 70% etoh for 1 min or store in 70% etoh up to a week at 4C


Jonathan's protocol[edit]

  • 100% confluence, and could wait one more day, and split
  • When cells are transferred from 6 well plate
    • 500uL of Trypsin, incubate 5min
    • Add 500uL media, and mix well, and
    • 100uL media in 96 well, or 300~350uL media in glass bottom dish
    • Add 60uL of cell in 96 well or 200uL in glass bottom dish
    • Culture one or two more day


  • Fixed cells just using 10% Formamide
    • Aspirate media
    • Add 50uL of 10% Formamide (200uL for glass bottom)
    • Incubate for 15min
    • Wash with PBS three times
    • Store in Ethanol


PGP1F cell culture in 96 well plate[edit]

  • Add 1mL Trypsin in T25 flask (about 90~100% covered)
  • Add 4mL media (total 5mL)
  • Add 100uL cell (from 5mL in T25 flask) to each well in 96 well plate (total 16 wells)
  • Add 200uL media to each well, and gently mix