Modified myotube prep

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.

Materials

• 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 6cm 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

Execution

1 Put 3.3mls SF MEM into the 5ml tube add 1 tube of fibronectin , mix (thaw fibronectin on ice) 2 (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

3 Incubate at RT for at least 2 hours ( or overnight at 4 degrees C ) 4 Label the universal tubes:2 with “100um”, 2 with “35 um” and 1 with” myotubes” 5 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 ) 6 Put 9 mls low serum media in universal tube “ myotube”

and 2 mls low serum media in the 6 cm plate		

7 Check myotubes on microscope 8 Take 2 X10 cm plate of myotubes and aspirate off the media 9 Rinse the myotubes with 5mls APBS (aspirate) 10 Add 2mlTE to each plate-trypsinize very briefly until chunks of cells start coming off 11 Quickly stop TE with 6mls low serum media 12 Detach cells from plate, using pipette 13 Slowly pipette the liquid through the 100 um filter, rinse with 2 mls low serum media ( do it with both tubes ) 14 Remove 100 um filters from the tubes 15 Drip a small volume of L. S. media through the 35 um filter 16 Pipette the filtrate through the 35um filter and wash with 2 mls of low serum media 17 Place filter face down in a 6cm dish 18 Wash the filter with 2mls low serum( and then discard the filter),

19 Repeat steps 14-16 with the other tube 20 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” 21 Aspirate off the fibronectin from the 96 well-plate 22 Cut off tip of 5 ml pipette 23 ( Using multipipette) put 150 ul per well from “ myotube” into each well of the 96 well plate 24 Look in the microscope for myotubes 25 Label the 96 well-plate with name,date and

                          myotube in low serum ( L. S. )		

26 Put cells in incubator

Notes: (Date and passage number on the 10 cm dish)