TITLE: Human embryonic gonad dissociation with Collagenase & Trypsin
AUTHORS: Carmen Sancho, Regina Hoo, Roser Vento-Tormo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is for enrichment of fetal gonadal cells</div></div>

[STEPS]
?. [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
?. [Prepare collagenase mix]
Digestion mix recipe:ReagentsVolumeConcentration (stock)Trypsin-EDTA (0.25%) phenol red4ml (depending on tissue size)0.25% (1X)DNaseI (0.1mg/ml)40ul10mg/ml
ReagentsVolumeConcentration (stock)Trypsin-EDTA (0.25%) phenol red4ml (depending on tissue size)0.25% (1X)DNaseI (0.1mg/ml)40ul10mg/ml
?. [Tissue dissociation and digestion]
Wash tissue with PBS.
?. [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 15ml 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]
Filter sample through small strain (100 um) and keep the filter.
?. [Tissue dissociation and digestion]
Centrifuge 450 g,  (0.5 rcf, 5mins), 4 degrees
?. [Tissue dissociation and digestion]
Remove carefully 90% of the media by decanting the tube (leave between 0.5 ml and 1 ml).
?. [Tissue dissociation and digestion]
Resuspend the cell pellet in 2 ml of PBS and pass it through the inverted filter adding extra 3 mL of PBS
?. [Tissue dissociation and digestion]
Centrifuge 450g, 5 min, 4 degrees
?. [Tissue dissociation and digestion]
Remove carefully 90% of the media by decanting the tube (leave between 0.5 ml and 1 ml) and transfer to a 1mL eppendorf
?. [Tissue dissociation and digestion]
Centrifuge 450 g,  (0.5 rcf, 5mins) and discard supernatant
?. [Tissue dissociation and digestion]
Add 700 microL Trypsin-EDTA/DNase I and Incubate at  for 10 min with rotation.
37 °C
?. [Tissue dissociation and digestion]
Add 700 microL RPMI complete and filter trhough a Facs tube passing 1mL more of media.
?. [Tissue dissociation and digestion]
Transfer to a 5 mL eppendorf and Centrifuge 450 g,  (0.5 rcf, 5mins)
?. [Tissue dissociation and digestion]
Discard supernatant without disrupting the cell pellet and with 1 mL of PBS-BSA 0.04%, transfer to a 1,5 mL eppendorf
?. [Tissue dissociation and digestion]
Centrifuge 450 g,  (0.5 rcf, 5mins) and leave 200-300 microL
?. [Tissue dissociation and digestion]
Proceed to cell count with disposable C-chip haematocytometer.