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

Published: May 31, 2024

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 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% p…

Representative 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 Acquisition | Auto-save to… (B) Overview of the imaging parameters. Once the mouse has been pos…

Divulgazioni

The authors have nothing to disclose.

Materials

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

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Citazione di questo articolo
Imaging Transgenic Beta-Cells in an Immunodeficient Mouse Challenged with Diabetic Splenocytes. J. Vis. Exp. (Pending Publication), e22278, doi: (2024).

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