Retro-Orbital Blood Sampling: A Method for Isolating Mononuclear Cells from the Retro-Orbital Sinus of a Mouse

Abstract

Source: Mopin A. et al. A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model. J. Vis. Exp. (2016)

The video describes a sampling method for peripheral blood collection from retro-orbital sinus of a mouse and further isolation of mononuclear cells from blood with density gradient centrifugation.

Protocol

1. Retro orbital blood collection

1. Perform retro orbital blood collection just before euthanasia under sterile conditions in a laminar flow hood and under a heating lamp to prevent hypothermia.
2. For anesthesia, use ketamine at 150 mg/kg and xylazine at 10 mg/kg. Prepare the anesthetic solution by diluting 1.5 ml of ketamine and 0.5 ml of xylazine in 18 ml of PBS solution.
3. Insert a capillary tube into the medial canthus of the eye. Blood will rise from the orbital sinus into the capillary tube. Control the bleeding by gently applying pressure onto the eye with a sterile gauze sponge.
   NOTE: A volume ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ketamine 1000 (100 mg/ml)	VIRBAC	3.59713E+12
Xylazine SEDAXYLAN (20 mg/ml)	CEVA
PBS solution (1x concentrate)	ThermoFisher Scientific	14190
Ultra Pure 0.5 M EDTA solution, pH 8.0	ThermoFisher Scientific	15575
Separating solution (Pancoll Mouse)	PANBIOTECH	P04-64100
EDTA Tube	Greiner Bio-One	454034
Pasteur Pipette 150 mm (capillary tube)	Fisherbrand	1154-6963
Flow cytometry tubes (blue)	Beckman Coulter	2523749
Microcentrifuge tube (1.5 ml)	ThermoFisher Scientific	05-408-129
Sterile gauze sponges	URGO	501580
30 G needle	Becton & Dickinson	304000
1 ml syringe	Terumo	SS-01T
Shandon Cytospin 3 Cytocentrifuge	ThermoFisher Scientific