Induction of Graft Versus Host Disease in Mouse Models via Allogeneic Bone Marrow and Splenic Cell Transplantation

Abstract

Source: Buchele, V., et al. Induction of Intestinal Graft-versus-host Disease and Its Mini-endoscopic Assessment in Live Mice. J. Vis. Exp. (2019).

This video demonstrates the induction of intestinal graft-versus-host disease, GvHD, in a lethally-irradiated recipient mouse using allogeneic hematopoietic stem cells and GvHD-inducing splenic alloreactive T lymphocytes. This protocol helps establish a severe acute intestinal GvHD model to understand the mechanism of the disease and develop innovative therapeutic options.

Protocol

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

1. GvHD induction

1. Day 0: Total body irradiation of the recipient mice
   1. Use female CD45.2+ H2kd+ BALB/c mice that are at least 10 weeks old as recipients.
   2. Weigh and record the weight of the recipient mice prior to GvHD induction.
      NOTE: Ensure that the recipient has a minimal body weight of 20 g. The starting body weight will serve as the reference value to calculate the weight loss of the in....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BIOBEAM 2000 Gamma Irradiator	Gamma-Service Meical GmbH
Phosphate-buffered saline (PBS )	Sigma-Aldrich Co. LLC.	D8662-6x500ML
Semken Forceps (lenght: 13 cm; serrated, curved tips)	Fine Science Tools	11009-13
Hardened Fine Scissors (lenght: 8,5 cm; straight tips; cutting edge: 24 mm)	Fine Science Tools	14090-09
RPMI-1640 Medium	Sigma-Aldrich Co. LLC.	R8758-500ML
Hypodermic needle (26G)	B. Braun Melsungen AG	4657683
1 mL syringe	B. Braun Melsungen AG	9166017V
50 mL tube	Sarstedt Ag & Co.KG	6,25,47,254
Cell strainer with a 40 µm mesh screen	BD Falcon	352340
Insulin syringe with sterile interior (30G)	BD	324826
Pan T Cell Isolation Kit II, mouse	Miltenyi Biotec GmbH	130-095-130	magnet cell separation to isolate splenic T cells
CD90.2 MicroBeads, mouse	Miltenyi Biotec GmbH	130-049-101	magnet cell separation to isolate T cell-depleted bone marrow cells
Biopsy Forceps	KARL STORZ SE &Co KG	61071 ZJ	part of the experimental setup for colonoscopy
Ethylenediaminetetraacetic acid disodium salt dihydrate (Na2EDTA)	Carl Roth GmbH & Co.KG	8043.2	ingredient of ACK lysing buffer
Alexa Fluor 700 anti-mouse CD45.2 Antibody (clone: 104; lot: B252126; RRID: AB_493731)	Biolegend	109822
PE/Cy7 anti-mouse CD45.1 Antibody (clone: A20; lot: B217246; RRID: AB_1134168)	Biolegend	110730
APC/Cy7 anti-mouse CD8a Antibody (clone: 53-6.7, lot: B247008; RRID: AB_312753)	Biolegend	100714
Forane / Isofluran	AbbVie Inc.	B506
Filtrated bovine serum	Pan Biotec	P40-47500	ingredient of FACS buffer