Activation of Mouse Bone Marrow-Derived Macrophages in Response to Antibodies

Abstract

Source: Kozicky, L. et al., Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation. J. Vis. Exp. (2017)

In this video, we demonstrate the activation of bone marrow-derived macrophages in response to bacterial lipopolysaccharide and intravenous immunoglobulin.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Bone Marrow Macrophage Derivation and Activation with Antibodies

1. Perform euthanasia using CO₂ asphyxiation.
   1. Place the mouse in the induction chamber. Euthanize the mouse with 5% isoflurane anesthesia, for 60 - 90 s, until immobile and breathing is deep and slow.
   2. Turn off isoflurane anesthesia and administer 6 - 8 L/min of CO₂ until the mouse has stopped breathing. Leave the mouse with CO₂ on for at least another....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Iscove's modified Dulbecco's medium (IMDM)	Life technologies	12440053
Fetal Bovine Serum (FBS)	Life technologies	12483-020
Recombinant murine macrophage colony-stimulating factor (MCSF)	Stemcell technologies	78059
Penicillin-streptomycin	Life technologies	15140148
Cell dissociation buffer	Life technologies	13150016	Enzyme-Free, Hanks's-based, EDTA
Lipopolysaccharide (LPS)	Sigma aldrich	L 4516	From E. coli 0127:B8
IVIg (Gammunex)	Grifols	Received from BC Children's Hospital, Transfusion Medicine
IVIg (Gammagard liquid)	Baxter Healthcare Corporation	Received from BC Children's Hospital, Transfusion Medicine
IVIg (Octagam)	Octapharma	Received from BC Children's Hospital, Transfusion Medicine
Phosphate-buffered saline (PBS) (sterile), pH 7.4	Life technologies	10010023
Mouse IL-10 ELISA	BD biosciences	555252
Mouse IL-12/23p40 ELISA	BD biosciences	555165
15 mL conical tube	BD biosciences	352096
50 mL conical tube	BD biosciences	352070
Microcentrifuge tube (1.7 mL)	Diamed	SPE155-N
75 cm2 tissue culture treated flask	BD biosciences	353136
Cell scraper	BD biosciences	353085