TITLE: Myometrium Single Cell Dissociation Protocol
AUTHORS: Nicole Ulrich, Yu-Chi Shen, Sue Hammoud

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the single cell dissociation of myometrial cells.</div><div class = "text-block"><span>The single cell dissociation protocol for cells from fallopian tubes can be found </span><a href="https://www.protocols.io/private/6F9908C5F50A11EAA77F0A58A9FEAC2A" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">here</span></a><span style = ":;">.</span></div></div>

[STEPS]
?. [Myometrium single cell dissociation]
Sample arrives in HBSS, at .
0 Room temperature
?. [Myometrium single cell dissociation]
Weigh and divide sample to -.
150 mg
[section of tissue]
?. [Myometrium single cell dissociation]
Make several cuts in thin sections in the tissue (similar to a bivalve for the fallopian tube) but do not “mince” (- per tube).
150 mg
[tissue]
?. [Myometrium single cell dissociation]
Place - in  and place on  shaker, , -.
150 mg
[tissue]
[pronase]
37 °C
33
?. [Myometrium single cell dissociation]
Filter cell suspension #1 using a 100 μm filter, remove tissue from filter, rinse filter with  (important for quenching digestion), spin filtrate at , resuspend the pellet in , place .
[DMEM/FBS]
Centrifuge: 600 34, 5 min
[DMEM/FBS]
on ice
?. [Myometrium single cell dissociation]
Re-suspend tissue in digestion buffer and place on  shaker, .
37 °C
33
?. [Myometrium single cell dissociation]
After  (60 min total digestion) filter cell suspension #3 as above.
?. [Myometrium single cell dissociation]
After  (80 min total digestion) filter cell suspension #4 as above.
?. [Myometrium single cell dissociation]
After  (100 minutes total digestion), filter cell suspension #5 as above, discarding remaining tissue after it is removed.
?. [Myometrium single cell dissociation]
Combine all cell suspensions from each step ( suspension from each sample,  total) in FACS tube.
100 µl
500 µl
?. [Myometrium single cell dissociation]
Filter cell suspension #2 using a 100 μm filter.
?. [Myometrium single cell dissociation]
Remove the tissue from the filter and re-suspend in digestion buffer. Place back on  shaker,  for another .
37 °C
33
?. [Myometrium single cell dissociation]
After tissue is removed, rinse the filter with  to quench.
[DMEM/FBS]
?. [Myometrium single cell dissociation]
Spin filtrate #2 at , resuspend the pellet in .
Centrifuge: 600 34, 5 min
[DMEM/FBS]
?. [Red Cell Removal]
Add .
[red cell solution]
?. [Red Cell Removal]
Vortex  and incubate  at .
370 Room temperature
?. [Red Cell Removal]
Centrifuge . Remove supernatant and discard.
Centrifuge: 600 34, 5 min
?. [Red Cell Removal]
Re-suspend pellet in  or can proceed to washes.
[DMEM]
?. [Washes]
Wash 3 times with  in FACS tube. Spin down at  between washes.
[PBS/0.04% BSA]
Centrifuge: 600 34, 5 min
?. [Washes]
Wash with  in FACS tube. (1/3)
[PBS/0.04% BSA]
?. [Washes]
Spin at . (1/2)
Centrifuge: 300 34, 3 min
?. [Washes]
Wash with  in FACS tube. (2/3)
[PBS/0.04% BSA]
?. [Washes]
Spin at . (2/2)
Centrifuge: 300 34, 3 min
?. [Washes]
Wash with  in FACS tube. (3/3)
[PBS/0.04% BSA]
?. [Washes]
Use 100 μm to filter after last wash.
?. [Washes]
Re-suspend pellet in .
[DMEM/10%FBS]
?. [Washes]
Add DAPI at 1:500 for flow cytometry analysis for live/dead.
?. [To make slides after sorting]
Apply  to the slide and allow slide to dry in the incubator for -.
[single cell solution]
?. [To make slides after sorting]
Draw wax circles.
?. [To make slides after sorting]
Fix with  x .
[PFA]
?. [To make slides after sorting]
Wash 2 x  in PBS, store at .
4 °C