TITLE: Fallopian Tube 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 cells from fallopian tubes.</div><div class = "text-block"><span>The single cell dissociation protocol for myometrial cells can be found </span><a href="https://www.protocols.io/private/41B5CB84F51411EAA77F0A58A9FEAC2A" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">here</span></a><span style = ":;">.</span></div></div>

[STEPS]
?. [Fallopian tube single cell dissociation]
Each anatomic segment arrives in a separate tube in HBSS, at .
0 Room temperature
?. [Fallopian tube single cell dissociation]
Weigh and bivalve each fallopian tube segment, but do not “mince” (- per tube).
100 mg
[tissue]
?. [Fallopian tube single cell dissociation]
Place only isthmus/ampulla tissue (not fimbria) in (- per tube) and place on  shaker, , -.
[pronase]
100 mg
[tissue]
37 °C
33
?. [Fallopian tube single cell dissociation]
Filter cell suspension #1 for isthmus and ampulla using a 70 μm filter, remove tissue and place in the digestion tube.
?. [Fallopian tube single cell dissociation]
Rinse filter with . This is important for quenching the digestion.
[DMEM/FBS]
?. [Fallopian tube single cell dissociation]
Spin filtrate #1 at , resuspend pellet in , place .
Centrifuge: 400 34, 4 min
[DMEM/FBS]
on ice
?. [Fallopian tube single cell dissociation]
Suspend bi-valved tubal segment tissue (all segments) in individual labeled tubes of digestion buffer. ( with -). Place on  shaker, .
[pre-warmed digestion buffer]
100 mg
[tissue]
37 °C
33
?. [Fallopian tube single cell dissociation]
After  strain cell suspension #2 using a 70 μm filter, remove remaining tissue from filter and place back in digestion tube.
?. [Fallopian tube single cell dissociation]
Rinse filter with  to quench.
[DMEM/FBS]
?. [Fallopian tube single cell dissociation]
Spin filtrate #2 at , resuspend pellet in , place .
Centrifuge: 400 34, 4 min
[DMEM/FBS]
on ice
?. [Fallopian tube single cell dissociation]
Repeat digest for an additional  with fresh digestion buffer from stock.
?. [Fallopian tube single cell dissociation]
After  (60 minutes total digestion) filter cell suspension #3 using a 70 μm filter.
?. [Fallopian tube single cell dissociation]
Rinse filter with  to quench. Spin the filtrate at , resuspend pellet in , place .
[DMEM/FBS]
Centrifuge: 400 34, 4 min
[DMEM/FBS]
on ice
?. [Fallopian tube single cell dissociation]
Combine cell suspensions  from supernatants 1-3 (ampulla and isthmus) or 2-3 (fimbria - nopronase step) in one FACS tube for each segment for a total of  for ampulla and isthmus, and  for fimbria.
on ice
300 µl
200 µl
?. [Red Cell Removal]
Add  per  in each tube for each anatomic section (fimbria, isthmus, ampulla).
[red cell solution]
100 µl
?. [Red Cell Removal]
Vortex  and incubate  at .
370 Room temperature
?. [Red Cell Removal]
Centrifuge . Remove supernatant and discard.
Centrifuge: 400 34, 4 min
?. [Red Cell Removal]
Re-suspend in  or can proceed to washes.
[DMEM]
?. [Washes]
Wash 3 times with  in a FACS tube. Spin at  in between washes.
[PBS/0.04% BSA]
Centrifuge: 300 34, 3 min
?. [Washes]
Use 40 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
?. [Washes]
Wash with  in a FACS tube. (1/3)
[PBS/0.04% BSA]
?. [Washes]
Spin at . (1/2)
Centrifuge: 300 34, 3 min
?. [Washes]
Wash with  in a FACS tube. (2/3)
[PBS/0.04% BSA]
?. [Washes]
Spin at . (2/2)
Centrifuge: 300 34, 3 min
?. [Washes]
Wash with  in a FACS tube. (3/3)
[PBS/0.04% BSA]