Imaging Transgenic Beta-Cells in an Immunodeficient Mouse Challenged with Diabetic Splenocytes

Abstract

Source: Stewart, T. et al., Bioluminescent Monitoring of Graft Survival in an Adoptive Transfer Model of Autoimmune Diabetes in Mice. J. Vis. Exp. (2022)

This video demonstrates an imaging-based method to visualize transplanted luciferase-expressing beta cells into a non-obese diabetic immunodeficient mouse. The introduction of splenocytes from a diabetic mouse leads to progressive beta cell destruction, visually confirmed by declining luminescence after substrate introduction.

Protocol

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

1. Engineering and maintenance of NIT-1 cell lines

1. Maintain NIT-1 cell lines, which share a genetic background with non-obese diabetic (NOD) mice, and 293FT cells in tissue culture-treated dishes in Dulbecco's Modified Eagle Medium (DMEM) containing 4.5 g/L glucose supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in a 37 °C incubator with 5% CO₂.
2. Grow the 293FT cells to 70%-80% confluence for transfection.
3. Per 1....

Results

Figure 1: Screenshots of the software commands for imaging bioluminescent grafts. (A) Prior to imaging, select Initialize to prepare the instrument. The images may be auto-saved to a folder of choice by selecting Ac.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.05% Trypsin, 0.53 mM EDTA	Corning	25-052-CI
293FT	Invitrogen	R70007	Fast-growing, highly transfectable clonal isolate derived from human embryonal kidney cells transformed with the SV40 large T antigen
ACK Lysing Buffer	Gibco	A10492-01
Alcohol prep pads, 70% Isopropyl alcohol	Amazon/Ever Ready First Aid	B08NWF31DX
BD 5ml Syringe Luer-Lok Tip	BD	309646
BD PrecisionGlide Needle 26G x 5/8 (0.45 mm x 16 mm) Sub-Q	BD	305115
BD 1 mL TB Syringe Slip Tip	BD	309659
Blasticidin S HCl	Corning	30-100-RB
Cell strainer premium SureStrain, 70 µm, sterile	Southern Labware	C4070	Or use similar sterile strainer with 40-70um pore size
CellDrop automated cell counter	Denovix	CellDrop BF-PAYG	Or use similar cell counter device
Corning 100 mL Penicillin-Streptomycin Solution, 100x	Corning	30-002-CI
Disposable Aspirating Pipets, Polystyrene, Sterile, Capacity=2 mL	VWR	414004-265	Or use similar aspirating pipette
D-Luciferin, Potassium Salt , Molecular Biology Grade, Powder, >99%	Goldbio	LUCK-100
DMEM, high glucose, pyruvate, no glutamine	Gibco	10313039
Falcon BD tubes, 50 mL	Fisher Scientific	14-959-49A
Fetal Bovine Serum	Gibco	10437-028
Forceps premium for tissues, 1 x 2 teeth 5 in, German Steel	Fisher Scientific	13-820-074
Glucose urine test strip	California Pet Pharmacy	u-tsg100	Or use similar test strip for glucose measurments in urine/blood
GlutaMAX–1 (100x)	Gibco	35050-061
Infrared heating lamp	Cole Parmer	03057-00	Or use similar infrared lamp
Insulin syringe 0.5 mL, U-100 29 G 0.5 in	Becton Dickinson	309306
Isoflurane, USP	Piramal Critical Care	6679401725
IVIS Spectrum in vivo imaging system	Perkin Elmer	124262	Instrument for non-invasively collecting bioluminescent images of transplanted cells
Living Image Analysis Software	Perkin Elmer	128113	Software for collecting and quantifying bioluminescent signal
Microcentrifuge tubes seal-rite, 1.5 mL	USA Scientific	1615-5510	Or use similar sterile microcentrifuge tubes
NIT-1	ATCC	CRL-2055	Pancreatic beta-celll line derived from NOD/Lt mice
NOD.Cg-Prkdcscid/J	The Jackson Laboratory	1303	Mice homozygous for the severe combined immune deficiency spontaneous mutation Prkdcscid, commonly referred to as scid, are characterized by an absence of functional T cells and B cells, lymphopenia, hypogammaglobulinemia, and a normal hematopoietic microenvironment.
NOD/ShiLtJ	The Jackson Laboratory	1976	The NOD/ShiLtJ strain of mice (commonly called NOD) is a polygenic model for autoimmune type 1 diabetes
PBS, pH 7.4	Thermo Fisher Scientific	10010031	No calcium, no magnesium, no phenol red
pCMV-VSV-G	Addgene	8454
pLenti-luciferase-blast	Made in-house	Plasmid available upon request	See Supplemental File 1
pMD2.G	Addgene	12259
pMDLg/pRRE	Addgene	12251
Polyethylenimine, Linear, MW 25,000, Transfection Grade (PEI 25K)	Fisher Scientific	NC1014320
pRSV-Rev	Addgene	12253
Restrainer for rodents, broome-style round 1 in	Fisher Scientific	01-288-32A
Scissors, sharp-pointed	Fisher Scientific	08-940	Or use other scissors made of surgical-grade stainless steel
Tissue-culture treated culture dishes	Millipore Sigma	CLS430167-20EA	Or use other sterile cell culture-treated Petri dishes
Tweezers/Forceps, fine precision medium tipped	Fisher Scientific	12-000-157