マウス膵島単離

Protocol

コラゲナーゼ準備下の表に示すように、最終濃度を、作るために50 mlコニカル及び追加の絶縁バッファにコラゲナーゼを秤量。ボルテックスによりコラゲナーゼを溶かす。 （12匹+ 2余分なマウス= 14マウス× 5 0.2ml /マウス= 70ミリリットルトータルコラゲナーゼ溶液） （70ミリリットル× 0.5 mg / mlまたはコラゲナーゼPの合計の0.3 mg / mlの= 35 mgまた?…

Materials

Material Name	Type	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