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

Published: April 30, 2023

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 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.

開示

The authors have nothing to disclose.

Materials

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

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記事を引用
Retro-Orbital Blood Sampling: A Method for Isolating Mononuclear Cells from the Retro-Orbital Sinus of a Mouse. J. Vis. Exp. (Pending Publication), e20263, doi: (2023).

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