Difference between revisions of "Modified myotube prep"
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(Created page with "Induce A1 cells on 10 cm plates to form myotubes by * wash 1x with 5 mls APBS * changing the media to 0.5% serum-AMEM * wait 4 days If desired, myotubes can be purified awa...") |
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If desired, myotubes can be purified away from contaminating mononucleate cells and replated using the following procedure. The yield from 2 x 10 cm plates of myotubes should be about 5,000-10,000 myotubes. Myotubes should be plated onto a fibronectin coated substratum. | If desired, myotubes can be purified away from contaminating mononucleate cells and replated using the following procedure. The yield from 2 x 10 cm plates of myotubes should be about 5,000-10,000 myotubes. Myotubes should be plated onto a fibronectin coated substratum. | ||
| − | + | == Material == | |
| − | + | * 1 x 0,5 % serum AEMEM (low serum media) | |
| − | + | * 2 x 100 um filters cut into 2 cm x 2 cm squares | |
| − | + | * 2 x 35 um filters | |
| − | + | * 1 x 6 cm plate containing 2mls of low serum media (0,5% serum AEMEM) | |
| − | + | * 1 x 96 well plate coated with 20 µg/ml fibronectin in SF media | |
| − | + | * 1 x scalpel number 22 | |
| − | + | * 1 x tweezers | |
| − | + | * 5 x universal (25 ml) tubes, | |
| − | + | * 1 x TE (trypsin – edta ) | |
| − | + | * 1 x APBS (100 ml PBS + 25 ml ddH2O | |
| − | + | * sterile pipettes(2 * 5mls, 2 * 10mls, 1 * 20mls) | |
| − | + | * sterile pasteur pipettes unplugged | |
| − | drawer number 3 ) | + | * 2 x 60 ul of fibronectin at 1mg/ml (in –20 degrees C freezer, drawer number 3 ) |
| − | + | * 1 x 4 ml tube | |
| − | + | * 500ul multipipette tip | |
| − | + | * 5ul multipipette tip | |
| − | + | * multipipette plus | |
| − | + | * microscope | |
| − | + | == Procedure == | |
| − | + | # Put 3.3mls SF MEM into the 5ml tube add 1 tube of fibronectin , mix (thaw fibronectin on ice) | |
| − | + | # (Using multipipette 500ul) put 30ul of fibronectin mix in each well of the 96 well plate, distribute the liquid so that it wets the bottom evenly | |
| − | + | # Incubate at RT for at least 2 hours ( or overnight at 4 degrees C ) | |
| − | + | # Label the universal tubes:2 with “100um”, 2 with “35 um” and 1 with” myotubes” | |
| − | wets the bottom evenly | + | # Put filters on the tubes and fix at 3 points with a hot scalpel ( leave the scalpel for 20 seconds in the flame, wait 8 sec., then use hot blade to melt filter onto the top of the tube ) |
| − | + | # Put 9 mls low serum media in universal tube “ myotube” and 2 mls low serum media in the 6 cm plate | |
| − | + | # Check myotubes on microscope | |
| − | + | # Take 2 X10 cm plate of myotubes and aspirate off the media | |
| − | + | # Rinse the myotubes with 5mls APBS (aspirate) | |
| − | and 1 with” myotubes” | + | # Add 2mlTE to each plate-trypsinize very briefly until chunks of cells start coming off |
| − | + | # Quickly stop TE with 6mls low serum media | |
| − | scalpel ( leave the scalpel for 20 seconds in the flame, wait 8 sec., then use hot blade to melt filter onto the top of the tube ) | + | # Detach cells from plate, using pipette |
| − | + | # Slowly pipette the liquid through the 100 um filter, rinse with 2 mls low serum media (do it with both tube) | |
| − | + | # Remove 100 um filters from the tubes | |
| − | + | # Drip a small volume of L. S. media through the 35 um filter | |
| − | + | # Pipette the filtrate through the 35um filter and wash with 2 mls of low serum media | |
| − | + | # Place filter face down in a 6cm dish | |
| − | + | # Wash the filter with 2mls low serum (and then discard the filter) | |
| − | + | # Repeat steps 14-16 with the other tube | |
| − | + | # Then pipette the liquid from the 6cm plate and put it into the myotube tube , wash the plate with some low serum media from the “ myotube” | |
| − | + | # Aspirate off the fibronectin from the 96 well-plate | |
| − | with 2 mls low serum media | + | # Cut off tip of 5 ml pipette |
| − | + | # (Using multipipette) put 150 ul per well from “ myotube” into each well of the 96 well plate | |
| − | + | # Look in the microscope for myotubes | |
| − | + | # Label the 96 well-plate with name, date and myotube in low serum ( L. S. ) | |
| − | + | # Put cells in incubator | |
| − | |||
| − | the filter) | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | the myotube tube , wash the plate with some low serum | ||
| − | media from the “ myotube” | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | Notes: | + | ''Notes:'' |
(Date and passage number on the 10 cm dish) | (Date and passage number on the 10 cm dish) | ||
Latest revision as of 14:24, 8 June 2015
Induce A1 cells on 10 cm plates to form myotubes by
- wash 1x with 5 mls APBS
- changing the media to 0.5% serum-AMEM
- wait 4 days
If desired, myotubes can be purified away from contaminating mononucleate cells and replated using the following procedure. The yield from 2 x 10 cm plates of myotubes should be about 5,000-10,000 myotubes. Myotubes should be plated onto a fibronectin coated substratum.
Material
- 1 x 0,5 % serum AEMEM (low serum media)
- 2 x 100 um filters cut into 2 cm x 2 cm squares
- 2 x 35 um filters
- 1 x 6 cm plate containing 2mls of low serum media (0,5% serum AEMEM)
- 1 x 96 well plate coated with 20 µg/ml fibronectin in SF media
- 1 x scalpel number 22
- 1 x tweezers
- 5 x universal (25 ml) tubes,
- 1 x TE (trypsin – edta )
- 1 x APBS (100 ml PBS + 25 ml ddH2O
- sterile pipettes(2 * 5mls, 2 * 10mls, 1 * 20mls)
- sterile pasteur pipettes unplugged
- 2 x 60 ul of fibronectin at 1mg/ml (in –20 degrees C freezer, drawer number 3 )
- 1 x 4 ml tube
- 500ul multipipette tip
- 5ul multipipette tip
- multipipette plus
- microscope
Procedure
- Put 3.3mls SF MEM into the 5ml tube add 1 tube of fibronectin , mix (thaw fibronectin on ice)
- (Using multipipette 500ul) put 30ul of fibronectin mix in each well of the 96 well plate, distribute the liquid so that it wets the bottom evenly
- Incubate at RT for at least 2 hours ( or overnight at 4 degrees C )
- Label the universal tubes:2 with “100um”, 2 with “35 um” and 1 with” myotubes”
- Put filters on the tubes and fix at 3 points with a hot scalpel ( leave the scalpel for 20 seconds in the flame, wait 8 sec., then use hot blade to melt filter onto the top of the tube )
- Put 9 mls low serum media in universal tube “ myotube” and 2 mls low serum media in the 6 cm plate
- Check myotubes on microscope
- Take 2 X10 cm plate of myotubes and aspirate off the media
- Rinse the myotubes with 5mls APBS (aspirate)
- Add 2mlTE to each plate-trypsinize very briefly until chunks of cells start coming off
- Quickly stop TE with 6mls low serum media
- Detach cells from plate, using pipette
- Slowly pipette the liquid through the 100 um filter, rinse with 2 mls low serum media (do it with both tube)
- Remove 100 um filters from the tubes
- Drip a small volume of L. S. media through the 35 um filter
- Pipette the filtrate through the 35um filter and wash with 2 mls of low serum media
- Place filter face down in a 6cm dish
- Wash the filter with 2mls low serum (and then discard the filter)
- Repeat steps 14-16 with the other tube
- Then pipette the liquid from the 6cm plate and put it into the myotube tube , wash the plate with some low serum media from the “ myotube”
- Aspirate off the fibronectin from the 96 well-plate
- Cut off tip of 5 ml pipette
- (Using multipipette) put 150 ul per well from “ myotube” into each well of the 96 well plate
- Look in the microscope for myotubes
- Label the 96 well-plate with name, date and myotube in low serum ( L. S. )
- Put cells in incubator
Notes: (Date and passage number on the 10 cm dish)