Near-Infrared Photoimmunotherapy for Targeted Cell Elimination in Mixed 3D Cultures
Published: April 30, 2024
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) Prepare mAb of interest at 2-5 mg/ml in 0.1 M disodium hydrogen phosphate (Na2HPO4) (pH 8.6) solution. Mix 6.8 nmol of mAb w…
Representative 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 (A431-luc-GFP) with no harm to non-target cells (3T3-RFP), in a mixed 3D spheroid. Bar = 200 µm. (C) Quantification of luci…
Declarações
The authors have nothing to disclose.
Materials
| 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 |
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Near-Infrared Photoimmunotherapy for Targeted Cell Elimination in Mixed 3D Cultures. J. Vis. Exp. (Pending Publication), e22226, doi: (2024).