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

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 of 100 to 200 µl of blood can be collected using this technique.
4. Collect the blood into an EDTA tube, and store the sample on ice before isolating the mononuclear cells.

2. Isolation of blood mononuclear cells

1. Transfer the blood sample (100 to 200 µl; obtained from step 1.1) into a microcentrifuge tube, and add PBS/1 mM EDTA solution until the solution volume is 500 µl. Carefully layer 500 µl of separating solution under the solution containing the blood using a 30G needle and a 1 ml syringe. Do not mix the blood and the separating solution.
2. Centrifuge the tubes at 800 x g (without brake) for 20 min at RT. After centrifugation, collect the cellular ring (the opaque white layer) using a pipette. Transfer the cells to a microcentrifuge tube.
   NOTE: The opaque white layer contains lymphocytes as well as monocytes and appears between the lower layer - the separating solution - and the upper layer.
3. Add 1 ml of PBS solution, and centrifuge the tube at 350 x g for 10 min. Resuspend the cells in 600 µl of FACS buffer.

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Materials

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