TITLE: Ovary tissue dissociation
AUTHORS: Regina Hoo, Carmen Sancho, Roser Vento-Tormo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Single cell suspension from ovary tissue and digestion of oocytes</div></div>

[STEPS]
?. [Tissue dissociation and digestion]
Wash tissue with PBS.
?. [Prepare collagenase mix]
Heat-inactivate FBS at  for  before use.Collagenase mix recipe:  ABCD1ProductStockFinal volume (10 ml)Concentration2RPMI or Hams F12
+ 10% FBS9 ml RPMI or Hams F12
+ 1ml FBS8.8 ml3Collagenase IA10 mg/ml1 ml1 mg/ml4Liberase TM5 mg/ml100 ul50 ug/ml5DNase I10 mg/ml100 ul0.1 mg/ml
56 °C
ABCD1ProductStockFinal volume (10 ml)Concentration2RPMI or Hams F12
+ 10% FBS9 ml RPMI or Hams F12
+ 1ml FBS8.8 ml3Collagenase IA10 mg/ml1 ml1 mg/ml4Liberase TM5 mg/ml100 ul50 ug/ml5DNase I10 mg/ml100 ul0.1 mg/ml
?. [Tissue dissociation and digestion]
Place wet tissue under a petri dish. Take 2 scalpels and roughly mince up the tissue. This step is crucial to increase the efficiency of the digestion.
?. [Tissue dissociation and digestion]
Transfer contents to 50ml falcon containing the collagenase mix (~/tissue but it will depend on the size of the tissue.
10 mL
?. [Tissue dissociation and digestion]
Tighten lid and then seal with parafilm.
?. [Tissue dissociation and digestion]
Incubate at for . Shacking every 10 min during the incubation is recommended.
37 °C
?. [Tissue dissociation and digestion]
Centrifuge 450 g,  (0.5 rcf, 5mins).
?. [Tissue dissociation and digestion]
Remove carefully 90% of the media by decanting the tube (leave between 0.5 ml and 1 ml).
?. [**If collecting for oocytes]
Filtered material: add ~ of RPMI 10%.
5 mL
?. [**If collecting for oocytes]
Transfer oocytes manually using high resolution microscope into drops of oocyte media.
?. [**If collecting for oocytes]
Clean oocytes.
?. [**If collecting for oocytes]
Options:A) Trypsin-EDTA (0.25%) phenol red (cat. #: 25200072, LifeTech).B) AccutaseC) Collagenase Mix
?. [**If collecting for oocytes]
Transfer oocytes and granulosa cells into  of lysis buffer in a PCR tube.
10 µl
?. [**If collecting for oocytes]
Centrifuge the other non-oocyte cells at 450 g,  (0.5 rcf, 5mins)
?. [**If collecting for oocytes]
Cells are resuspended in 2 ml of freezing media and aliquot in 2 cryopreservation tubes (1ml/each). Refer to "Cryopreservation of single cell suspension from tissue" protocol.
?. [Prepare oocyte lysis buffer ]
ABCD1ProductStockFinal volume (1 ml)Concentration2TCL lysis buffer990 ul3beta-mercaptoethanol10 ul1%
ABCD1ProductStockFinal volume (1 ml)Concentration2TCL lysis buffer990 ul3beta-mercaptoethanol10 ul1%
?. [Tissue dissociation and digestion]
Filter sample through small strain (100 um) and discard the filter. *Note: If collecting oocytes, proceed to step 18.
?. [**If collecting for oocytes]
Filter sample through small strain (100 um) – do not discard retained tissue that will be put it back in RPMI 10% and observed under the microscope.
?. [Tissue dissociation and digestion]
Resuspend the cell pellet in 5 ml of PBS.
?. [Tissue dissociation and digestion]
Check cell viability with trypan blue staining and on a with disposable C-chip haematocytometer. If cells is viable proceed to next step.
?. [Tissue dissociation and digestion]
Centrifuge 450 g for 5min. Discard supernatant.
?. [Tissue dissociation and digestion]
Resuspend the cell pellet in 5 ml of PBS. Centrifuge 450 g for 5min. Discard supernatant.
?. [Tissue dissociation and digestion]
Resuspend cells with 5ml of PBS. Proceed to cell count with disposable C-chip haematocytometer.
?. [Tissue dissociation and digestion]
Re-count cells to obtain accurate cell suspension concentration for loading 10X instrument.
?. [Tissue dissociation and digestion]
Freeze down excess cells, re-spin down any remaining cells, at 4°C, 450g, 5mins. Proceed to "Cryopreservation of single cell suspension from tissue"