Reconstituting Dissociation Reagents
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Dissociation Reagents Index
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Collagenase B (cat # 11 08 831 001) 2.5gms
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Collagenase (0.2%/20% FCS/1xPBS) from C. Histolyticum 1
(Cat # C-0130-Sigma) 5gms
Dispase (Stem Cell Technologies)
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DNase
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Trypsin-EDTA (Home made)
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Trypsin (Invitrogen, Cat #
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COLLAGENASE B (Cat # 11 08 831 001) 2.5gms
 Used to dissociate hES cells from MEFs/Matrigel.
 Want final concentration to be 1mg/ml.
 Weigh 500ml or 1 liter plastic bottle. Zero the weight of the bottle.
 In the fume hood, transfer some collagenase B to the bottle (500 ug- 1 mg).
 Weigh the bottle + collagenase B again and repeat process till you have 500ug- 1
mg of collagenase B in bottle.
 Add DMEM/F12 supplemented with glutamine and P/S so that you have a
concentration of 1mg/ml.
 Let dissolve at RT for 1 hour
 Filter and aliquot 12 ml in14 ml tubes.
 Freeze at –20.
COLLAGENASE (0.2%/20% FCS/1XPBS) from C. Histolyticum (Cat # C-0130Sigma) 5gms
 To be used to break up late stage EBs and embryos during MEF preparation.
 Weigh 500ml or 1 liter plastic bottle. Zero weight of bottle.
 In the fume hood, transfer some collagenase to the bottle (want approx 1 gm).
 Weigh the bottle + collagenase again and repeat process till you have 1gm of
collagenase in bottle.
 Add 400ml of 1X PBS + 100ml of Atlas FCS + 5ml of Glutamine + 5ml of P/S.
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 Let dissolve at 37 degrees for 15 minutes and shake it at intervals of 5 minutes.
 Filter and aliquot 12 ml in14 ml tubes.
 Freeze at –20.
DISPASE (Stem Cell Technologies, Cat # Catalog #07923 – 100ml)
Already reconstituted and no need to do anything.
DNASE (VWR, Cat # 80510-412, 10MU)
 Want final concentration to be 1mg/ml
 10MU X 1X106 U X 1mg
1 MU
65150 U
= 153mg
 In the hood transfer powder to a 125 ml bottle
 Bring the volume up to 153 ml with ice cold sterile water
 Let dissolve on ice for 1-2 hours
 Filter and aliquot 1ml/eppendorf
 Store at –20.
TRYPSIN-EDTA
 Make 500ml at a time.
 -Add 1.25g trypsin powder to bottle with 500ml 1XPBS.
 -Add 1.1 ml of 0.5M EDTA to bottle.
 -Stir gently, and then warm in water bath at 37C for a few minutes while stirring
to dissolve, until solution is clear.
 -Filter-sterilize and aliquot into 125ml bottles. Label with contents and date.
TRYPSIN-EDTA, 0.25%, 6X100ml (VWR, Cat # 45000-664)
Already reconstituted and no need to do anything.
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