Murine eilandjes van Langerhans Isolatie

Protocol

Collagenase Voorbereiding Weeg de collagenase in een 50 ml conische en voeg isolatie buffer aan de uiteindelijke concentratie, zoals aangegeven in de onderstaande tabel te maken. Los van de collagenase door vortexen. (12 muizen + 2 extra muizen = 14 muizen x 5 ml / muis = 70 ml in totaal collagenase oplossing) (70 ml x 0,5 mg / ml of 0,3 mg / ml = 35 mg of 21 mg collagenase P totaal) <table style="BORDER-RIGHT: #ddd 1px solid; BORDER-TOP:…

Materials

Material Name	Tipo	Company	Catalogue Number	Comment
Dithizone	reagent			Dissolve 5mg Dithizone in 5 ml of DMSO in a 50 ml conical tube. Add 45 ml of normal saline and mix gently. Filter with a 60 cc syringe and a 0.22 “m syringe filter into a new 50 ml tube. Store at 4 OC, until use. Add 1 ml to each 35 mm counting Grid-plate, when needed.
PBS	Buffer
HBSS	Buffer
Isolation soln.	Buffer			HBSS containing 10 mM of HEPES, 1 mM of MgCl2, 5 mM of Glucose, pH 7.4
Wash soln.	Buffer			Isolation buffer containing 1mM CaCl2
Collagenase P		Boehringer Mannheim-Roche	1213873
Dissection tools				Hemostat clamp (n=2), Forceps (n=2), Scissors (for skin incision an for taking out pancreas from adjacent tissue)
Syringe				sterile, 5ml, 1/mouse.
30 G needles				sterile, 1/syringe
siliconized glass vials w/Teflon cap		Fisher	213-018-54	sterile, 1/mouse
large sink strainer				sterile
1000 ml Beaker				sterile
Gauze pads				4×4, 2/mouse
plastic eye dropper				sterile
Mice	Animal