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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)
<ol>
	<li>Prepare mAb of interest at 2-5 mg/ml in 0.1 M disodium hydrogen phosphate (Na2HPO4)&#160;(pH 8.6) solution.</li>
	<li>Mix 6.8 nmol of mAb with 30.8 nmol of 10 mM IR700 in 0.1 M Na2HPO4&#160;solution (pH 8.6) in a microcentrifuge tube and incubate at RT for 1 hr, covered with aluminum foil.</li>
	<li>Wash the PD-10 column (see Table of Materials/Equipment) with 15 ml PBS, twice. Load the sample from step 1.2.</li>
	<li>Purify the mixture via the PD-10 column by PBS elution according to manufacturer&#39;s instructions.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: Here, the elution was along the band color of IR700. For a 300 &#181;l sample, PBS elution fraction is typically between 2.5 ml to 4.4 ml.</li>
	<li>Determine the protein concentration with Coomassie staining by measuring the absorption at 595 nm with a spectrophotometer. Determine the concentration of IR700 with absorption at 689 nm to confirm the number of fluorophore molecules conjugated to each mAb molecule.&#160;&#160;&#160;&#160;&#160; 
	NOTE: It is important to determine an optimal conjugation number of IR700 molecules in 1 mAb with a spectrophotometer. Generally, around 3 IR700 molecules in 1 mAb molecule is optimal for both&#160;in vitro&#160;and&#160;in vivo&#160;work. High-performance liquid chromatography (HPLC) and sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) methods can be used to confirm whether the mAb and IR700 are bound or not.</li>
	<li>Store at 4 &#176;C after determining the protein concentration.</li>
</ol>
2. Preparation of Mixed 3D Cell Culture (Mixed Spheroid)
<ol>
	<li>Apply sterile water (around 1 ml) into the plate reservoir section of hanging drop plates.</li>
	<li>Prepare various ratios of the cell types of interest &#8212; A431-luciferase-green fluorescent protein (A431-luc-GFP) cells and 3T3-red fluorescent protein (3T3-RFP) cells &#8212; with each sample containing 5,000 cells total, suspended in 50 &#181;l of culture media.&#160;&#160;&#160;&#160;&#160; 
	NOTE: Prepare 1:100, 10: 100, 25:100, 50:100,&#160;etc.&#160;ratios of cells types of interest in culture media depending on the cell-type.</li>
	<li>Incubate the mix for 5-7 days in the 96 well hanging drop plate in a humidified incubator at 37 &#176;C and 5% carbon dioxide. Change the culture media every 2 days. NOTE: To make the 3D spheroid precisely, handle the plate gently, as drops containing cells fall off easily before forming 3D shapes.</li>
	<li>Observe the morphology and size of the spheroids using an inverted brightfield microscope at 10X - 40X magnification. NOTE: Although it depends on cell types and the size of hanging-drop, ensure that the diameter of the spheroid is around 400-600 &#181;m after 7 days of incubation in the 96 well hanging drop plate.</li>
</ol>
3.&#160;In Vitro&#160;NIR-PIT for Mixed 3D Cell Culture
<ol>
	<li>Change the media of the hanging drop plates to 10 &#181;g/ml antibody-photoabsorber conjugate (APC) containing media, and incubate for 6 hr in a humidified incubator at 37 &#176;C and 5% carbon dioxide.</li>
	<li>After 6 hr incubation, wash the spheroid twice with fresh culture media (phenol red free). Gently transfer the spheroid to a glass-bottomed 50 mm dish with 100 &#181;l fresh phenol red free culture media using a sterile 200 &#181;l pipette tip with the tip cut off. Place one spheroid in each dish.</li>
	<li>Observe the spheroid with an inverted brightfield microscope to detect the change of morphology. To observe the optical reporters (e.g.,&#160;GFP and RFP) use fluorescence microscope with the following filter settings: GFP&#160;&#8212; 469 nm excitation filter, and 525 nm emission filter; RFP&#160;&#8212; 559 nm excitation filter, and 630 nm emission filter.</li>
	<li>Place the light-emitting diode (LED) above the glass-bottomed dish for irradiation. 
	NOTE: Spheroids can be exposed to NIR light either on the microscope or in the laminar hood.
	<ol>
		<li>Measure the power density of the NIR-light with an optical power meter. According to this measurement, irradiate NIR-light via light-emitting diodes (LEDs) which emit light at wavelengths of 670 to 710 nm at 2 J/cm2. 
		NOTE: LED light can penetrate maximum depth of approximately 5 inches. Cytotoxic effects of NIR-PIT is dependent only on given energy regardless of power density and duration of exposure.</li>
	</ol>
	</li>
	<li>After irradiation, transfer the spheroid into a new hanging drop plate with 50 &#181;l of fresh culture media and incubate for 1 day in a humidified incubator at 37 &#176;C and 5% carbon dioxide.</li>
	<li>Gently transfer the spheroid to a glass-bottomed dish with 100 &#181;l of fresh culture media (phenol red free) using a sterile 200 &#181;l pipette tip with the tip cut off. Observe the spheroid with a fluorescence microscope 1 day after NIR-PIT using filter settings described in step 3.3.
	<ol>
		<li>Detect dead cells by adding propidium iodide to the media at a final concentration of 2 &#181;g/ml, or by loss of cytoplasmic GFP-fluorescence using a fluorescence microscope (Figure 1B).</li>
	</ol>
	</li>
	<li>Repeat steps 3.1-3.6 if target cells are not completely eliminated.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>IRDye 700DX Ester Infrared Dye</td>
			<td>LI-COR Bioscience (Lincoln, NE, USA)</td>
			<td>929-70011</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>SIGMA-ALDRICH (St. Louis, MO, USA)</td>
			<td>S9763</td>
			<td></td>
		</tr>
		<tr>
			<td>Sephadex G25 column (PD-10)&#160;</td>
			<td>GE Healthcare (Piscataway, NJ, USA)</td>
			<td>17-0851-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie (bradford) Plus protein assay</td>
			<td>Thermo Fisher Scientific Inc (Waltham, MA, USA)</td>
			<td>PI-23200</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfecta3D 96-Well hanging Drop Plates</td>
			<td>3D Biomatrix Inc (Ann Arbor, MI, USA)</td>
			<td>HDP1096-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical power meter</td>
			<td>Thorlabs (Newton, NJ, USA)</td>
			<td>PM100</td>
			<td></td>
		</tr>
		<tr>
			<td>LED: L690-66-60</td>
			<td>Marubeni America Co. (Santa Clara, CA, USA)</td>
			<td>L690-66-60</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectibix (panitumumab)</td>
			<td>Amgen (Thousand Oaks, CA, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>35mm glass bottom dish, dish size 35mm, well size 10mm</td>
			<td>Cellvis (Mountain View, CA, USA)</td>
			<td>D35-10-0-N</td>
			<td></td>
		</tr>
	</tbody>
</table>

near-infrared photoimmunotherapy for targeted cell elimination in mixed 3d cultures

Source: Sato, K. et al., <a href="https://app.jove.com/v/53633">Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique.</a>&#160;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.

<img alt="Figure 1" src="/files/ftp_upload/22226/22226_Figure1.jpg" /> 
Figure 1. Target cell elimination in 3D cell spheroids (mixed spheroids of A431-luc-GFP and 3T3-RFP cells).&#160;(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 &#181;m. (C) Quantification of luci...

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

NOTE: The following protocol describes the necessary steps to eliminate specific cells using Near infrared photoimmunotherapy (NIR-PIT). Controls and ...

Take a culture of hanging drop mixed spheroids, which are heterogeneous cell aggregates consisting of target cells expressing growth factor receptors and a fluorescent protein, while non-target cells express a different-colored fluorescent protein.
Replace media with an antibody-photoabsorber conjugate comprising a target cell-specific monoclonal antibody and a photoabsorber dye.
Incubate to facilitate antibody binding to target cell receptors.
Wash the spheroids with media to remove unbound conjugates, then transfer them to a glass-bottom dish with media for light irradiation.
Expose the spheroids to near-infrared light for photoimmunotherapy, triggering a photochemical reaction of the photoabsorber dye on target cells, causing rapid cellular membrane damage and cell death.
This process eliminates target cells without damaging non-target cells.
Transfer the spheroids to a fresh hanging drop plate. Add media and incubate for spheroid growth.
Under a fluorescence microscope, the absence of target cell fluorescence indicates effective cell elimination, while the presence of non-target cell fluorescence confirms the selective nature of photoimmunotherapy.

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78 Views. 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 t...

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

Synthesis of Gold Nanoparticle Integrated Photo-responsive Liposomes and Measurement of Their Microbubble Cavitation upon Pulse Laser Excitation

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control over the drug release. However, most of the relevant technologies are still in the development process and are unprocurable by clinics. Here, we describe a facile fabrication of these photo-responsive NPs with commercially available gold NPs and thermo-responsive liposomes. Calcein is used as a model drug to evaluate the encapsulation efficiency and the release kinetic profile upon heat/light stimulation. Finally, we show that this photo-triggered release is due to the membrane disruption caused by microbubble cavitation, which can be measured with hydrophone.

This protocol describes a simple preparation method for gold nanoparticle integrated photo-responsive liposomes with the commercially available materials. It also shows how to measure the microbubble cavitation process of the synthesized liposomes upon the treatment of pulsed laser.

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control ...

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Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological synapse, a stereotyped cell-cell junction that regulates T cell activation and directionally targets effector responses. To study these processes effectively, an imaging approach that is tailored to capturing fast, polarized responses is necessary. This protocol describes such a system, which is based on a photoactivatable peptide-major histocompatibility complex (pMHC) that is non-stimulatory until it is exposed to ultraviolet light. Targeted decaging of this reagent during videomicroscopy experiments enables precise spatiotemporal control of TCR activation and high-resolution monitoring of subsequent cellular responses by total internal reflection (TIRF) imaging. This approach is also compatible with genetic and pharmacological perturbation strategies. This allows for the assembly of well-defined molecular pathways that link TCR signaling to the formation of the polarized cytoskeletal structures that underlie the immunological synapse.

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Near Infrared Photoimmunotherapy for Mouse Models of Pleural Dissemination

The efficacy of photoimmunotherapy can be evaluated more accurately with an orthotopic mouse model than with a subcutaneous one. A pleural dissemination model can be used for the evaluation of treatment methods for intrathoracic diseases such as lung cancer or malignant pleural mesothelioma.
Near-infrared photoimmunotherapy (NIR-PIT) is a recently developed cancer treatment strategy that combines the specificity of tumor-targeting antibodies with toxicity caused by a photoabsorber (IR700Dye) after exposure to NIR light. The efficacy of NIR-PIT has been reported using various antibodies; however, only a few reports have shown the therapeutic effect of this strategy in an orthotopic model. In the present study, we demonstrate an example of efficacy evaluation of the pleural disseminated lung cancer model, which was treated using NIR-PIT.

Near-infrared photoimmunotherapy (NIR-PIT) is an emerging cancer therapeutic strategy that utilizes an antibody-photoabsorber (IR700Dye) conjugate and NIR light to destroy cancer cells. Here, we present a method to evaluate the antitumor effect of NIR-PIT in a mouse model of pleural disseminated lung cancer and malignant pleural mesothelioma using bioluminescence imaging.

The efficacy of photoimmunotherapy can be evaluated more accurately with an orthotopic mouse model than with a subcutaneous one. A pleural dissemination ...

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

Transcript

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

Synthesis of Gold Nanoparticle Integrated Photo-responsive Liposomes and Measurement of Their Microbubble Cavitation upon Pulse Laser Excitation

Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist

Near Infrared Photoimmunotherapy for Mouse Models of Pleural Dissemination