Hosuk:Cell fixation in culture dish: Difference between revisions
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>Hosuki78 (Created page with "#200uL of formalin (~39% formaldehyde)directly to 2 ml of culture dish, incubate for 15min at 37C #Change media of fresh formalin+PBS, incubate for 15min at 37C, #Wash with PB...") |
>Hosuki78 No edit summary |
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(6 intermediate revisions by the same user not shown) | |||
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# | *For cells in MatTek glass bottom dish(P/N : P35G-1.5-14-C) | ||
# | #NO aspirate media. add 200 ul formalin(~39% formaldehyde) to 2 ml media directly, | ||
# | #incubate for 15 min at 37C | ||
#aspirate | |||
#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)
- NO aspirate media. add 200 ul formalin(~39% formaldehyde) to 2 ml media directly,
- incubate for 15 min at 37C
- aspirate
- 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[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