In Vivo Activation of Peritoneal Macrophages in Response to Antibodies in a Murine Model

Abstract

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

This video demonstrates the activation of a mouse's peritoneal macrophages using intravenous immunoglobulin and bacterial lipopolysaccharide. The co-stimulation activates the peritoneal macrophages into anti-inflammatory macrophages possessing immune regulatory activity.

Protocol

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

1. Challenging Mice in Vivo with intravenous immunoglobulin and lipopolysaccharide (IVIg+LPS) and Harvesting Peritoneal Macrophages

1. Weigh each mouse. Calculate the volume of IVIg (100 mg/mL stock concentration) needed to provide a final dose of 2.5 g/kg body weight. Calculate the amount of LPS (100 μg/mL stock concentration in Iscove's modified Dulbecco's medium (IMDM)) needed to provide a final dose of 0.2 μg/g body weight.   ....

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
Penicillin-streptomycin	Life technologies	15140148
1X red blood cell lysis buffer	eBioscience
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
26 g needle	BD biosciences	305110
1 mL syringe	BD biosciences	309659
10 mL syringe	BD biosciences	309604
15 mL conical tube	BD biosciences	352096
Forceps	VWR	82027-386	Fine tip, dissecting
Scissors	VWR	82027-582	Delicate, 4 1/2"
Brightfield microscope	Motic	AE31	Inverted phase contrast
Scale	Mettler	PE 3000