Type 4 Collagenase Enzyme Mix (2 x 1 mL)
50 µL Type 4 Collagenase 100 mg/mL (5 mg/mL final conc.)
5 µL 1 M CaCl2 (5 mM final conc.)
10 µL 10% BSA/PBS (0.1% BSA final conc.)
Collagenase Type 4 - Worthington (LS004186) - 100 µL aliquots of 100 mg/mL, frozen at -80 °C
DNAse 1 - Applichem (A3778) – 10 µL aliquots each with 250 U, frozen at -80 °C
DPBS - ThermoFisher (cat. #14190)
Red Blood Cell Lysis Buffer - Sigma (R7757)
Trypan Blue Solution 0.4% - Gibco (15250061)
Required Equipment & Consumables:
Pipettes and pipet tips (MLS)
15, 50 ml Conicals (MLS)
1.5 mL tubes (MLS)
30 µM filters (MACS SmartStrainers, 130-098-458)
Petri dishes (MLS)
Razor blades (MLS)
Ice bucket w/ice (MLS)
Hemocytometers - InCyto Neubauer Improved (DHC-NO1-5)
The protocol workflow is as follows:
Isolate pancreas
First layer
Second layer
Preparing cells for Chromium/DropSeq