Matrigel atþ 4 C for 4 h. Just before use, dilute it at 1/10 in
cold DMEM/F-12 and keep on ice.- To dissolve Ultroser™G (Pall Corporation, #15950-017), add
20 ml of sterile H 2 O on the top of the powder without shaking.
After 20 min of incubation at RT, vigorously shake and wait
until complete dissolution. Pass it through a 0.22μm filter.
Add to the media or froze it until further use. - As concentration of the Ki67 antibody can differ between
batches, titration should be performed. - The stock solution is 2 mM (w/v) in water. Dilute the stock
solution in 1PBS to obtain a solution at 2 nM (v/v), and
keep it atþ 4 C for 1 week only. - Use one 30 ml container and one petri dishpermouse. If the
amount of muscle is less than the whole hind limbs (e.g., two
tibialis anterior), the use of 4 ml polypropylene FACS tubes for
the digestion step is recommended. - Mice have to be dissected in<5 min after death. If the time of
dissection is longer, the number of viable cells will decrease. - A too long incubation in the digestion buffer (more than 1 h)
will result in an increase of leaking labeling and of death cell. - Be careful the mixture is very dense and the cell strainer can be
blocked. Change the filter every time it is required. - For 1 ml of sample, take off 50μl to perform control tubes
(non-labeled, single labeling, and CD34-FITC FMO). Adjust
the volume in function of your sample. - Quantity of antibody is for one entire mouse. For the single
tubes (single labeling and FMO), use 100μl of the antibody
mix. - Viable cells do not incorporate the DAPI and are so negative
for it. - Due to side effect of the border that could perturb MuSC fate,
avoid the use of 96-well plate. - Plate the cells in a high volume (400–500μl/well) to avoid side
effect of the meniscus. - In this assay we do not make the distinction between Ki67 and
MyoD expression. To do so, change one of the two antibodies
for a goat-made antibody, for example. - Fusion is evaluated as the percentage of nuclei into myotubes
among the total number of nuclei. - For all the washes, carefully discard supernatant with a needle,
and put 1PBS with another needle on a 2.5 ml syringe. All
the volumes here are for 48-well plate.
548 Marine Theret et al.