Near-Infrared Photoimmunotherapy for Targeted Cell Elimination in Mixed 3D Cultures

Abstract

Source: Sato, K. et al., Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique. J. Vis. Exp. (2016)

This video demonstrates a technique for selectively removing specific cells from a mixed 3D cell culture through near-infrared photoimmunotherapy. In this method, targeted cells are accurately identified using an antibody conjugated with an NIR photoabsorber. Subsequent irradiation with infrared light induces cell death, effectively eliminating the targeted cells from the 3D culture.

Protocol

NOTE: The following protocol describes the necessary steps to eliminate specific cells using Near infrared photoimmunotherapy (NIR-PIT). Controls and other details about NIR-PIT and cell viability can be found elsewhere.

1. Conjugation of Infrared 700 (IR700) to Monoclonal Antibodies (mAb)

1. Prepare mAb of interest at 2-5 mg/ml in 0.1 M disodium hydrogen phosphate (Na₂HPO₄) (pH 8.6) solution.
2. Mix 6.8 nmol of mAb with 30.8 nmol of 10 mM IR700 in 0.1 M Na₂HPO₄ solution (pH 8.6) in a microcentrifuge tube and incubate at RT for 1 hr, covered with a....

Results

Figure 1. Target cell elimination in 3D cell spheroids (mixed spheroids of A431-luc-GFP and 3T3-RFP cells). (A) NIR-PIT (2 J/cm²) regimen is shown. (B) Repeated NIR-PIT completely eliminated target cells .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
IRDye 700DX Ester Infrared Dye	LI-COR Bioscience (Lincoln, NE, USA)	929-70011
Na2HPO4	SIGMA-ALDRICH (St. Louis, MO, USA)	S9763
Sephadex G25 column (PD-10)	GE Healthcare (Piscataway, NJ, USA)	17-0851-01
Coomassie (bradford) Plus protein assay	Thermo Fisher Scientific Inc (Waltham, MA, USA)	PI-23200
Perfecta3D 96-Well hanging Drop Plates	3D Biomatrix Inc (Ann Arbor, MI, USA)	HDP1096-8
Optical power meter	Thorlabs (Newton, NJ, USA)	PM100
LED: L690-66-60	Marubeni America Co. (Santa Clara, CA, USA)	L690-66-60
Vectibix (panitumumab)	Amgen (Thousand Oaks, CA, USA)
35mm glass bottom dish, dish size 35mm, well size 10mm	Cellvis (Mountain View, CA, USA)	D35-10-0-N