Name: ovarian cancer detection using photoacoustic flow cytometry
Uploaded: 2020-01-17T14:00:00
Description: Watch this Scientific Journal Video about Ovarian Cancer Detection Using Photoacoustic Flow Cytometry at JoVE.com

The authors would like to acknowledge Madeleine Howell for her help with synthesis, Matthew Chest for his help designing the flow system, and Ethan Marschall for assistance with SolidWorks.

1. Nanoparticle Synthesis and Functionalization
NOTE: Synthesis of the FA-CuS NPs is achieved using a one pot synthesis method adapted from a previously published protocol21. 
CAUTION: All synthesis should occur in a ventilated chemical fume hood.
<ol>
	<li>Prior to synthesis, filter approximately 300 mL of deionized (DI) water though a 0.2 &#181;m sterile filter.</li>
	<li>Clean a 250 mL glass round bottom flask with a detergent solution and rinse with DI water....

<ol>
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X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+value+of+HE4++circulating+tumor+cells+in+patients+with+suspicious+ovarian+cancer.">Diagnostic value of HE4+ circulating tumor cells in patients with suspicious ovarian cancer.</a> Oncotarget. 9 (7), 7522-7533 (2018).</li><li>Lianidou, E., Hoon, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=9+-+Circulating+Tumor+Cells+and+Circulating+Tumor+DNA.">9 - Circulating Tumor Cells and Circulating Tumor DNA.</a> Principles and Applications of Molecular Diagnostics. , 235-281 (2018).</li><li>Gorges, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumour+cells+escape+from+EpCAM-based+detection+due+to+epithelial-to-mesenchymal+transition.">Circulating tumour cells escape from EpCAM-based detection due to epithelial-to-mesenchymal transition.</a> BMC cancer. 12 (1), 178 (2012).</li><li>Galanzha, E., Zharov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cell+detection+and+capture+by+photoacoustic+flow+cytometry+in+vivo+and+ex+vivo.">Circulating tumor cell detection and capture by photoacoustic flow cytometry in vivo and ex vivo.</a> Cancers. 5 (4), 1691-1738 (2013).</li><li>Nedosekin, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+noninvasive+analysis+of+graphene+nanomaterial+pharmacokinetics+using+photoacoustic+flow+cytometry.">In vivo noninvasive analysis of graphene nanomaterial pharmacokinetics using photoacoustic flow cytometry.</a> Journal of Applied Toxicology. 37 (11), 1297-1304 (2017).</li><li>Zharov, V. P., Galanzha, E. I., Kim, J., Khlebtsov, N. G., Tuchin, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoacoustic+flow+cytometry:+principle+and+application+for+real-time+detection+of+circulating+single+nanoparticles,+pathogens,+and+contrast+dyes+in+vivo.">Photoacoustic flow cytometry: principle and application for real-time detection of circulating single nanoparticles, pathogens, and contrast dyes in vivo.</a> Journal of Biomedical Optic. 12 (5), 1-14 (2007).</li><li>Miranda, C., Sampath Kumar, S., Muthuswamy, J., Smith, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoacoustic+micropipette.">Photoacoustic micropipette.</a> Applied Physics Letters. 113 (26), 264103 (2018).</li><li>Miranda, C., Barkley, J., Smith, B. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capture+of+circulating+tumor+cells+using+photoacoustic+flowmetry+and+two+phase+flow.">Capture of circulating tumor cells using photoacoustic flowmetry and two phase flow.</a> Journal of Biomedical Optics. 17 (6), 061221 (2012).</li><li>Cai, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoacoustic+flow+cytometry+for+single+sickle+cell+detection+in+vitro+and+in+vivo.">Photoacoustic flow cytometry for single sickle cell detection in vitro and in vivo.</a> Analytical Cellular Pathology. 2016, 11 (2016).</li><li>Galanzha, E. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+magnetic+enrichment,+photoacoustic+diagnosis,+and+photothermal+purging+of+infected+blood+using+multifunctional+gold+and+magnetic+nanoparticles.">In vivo magnetic enrichment, photoacoustic diagnosis, and photothermal purging of infected blood using multifunctional gold and magnetic nanoparticles.</a> PLoS One. 7 (9), e45557 (2012).</li><li>Hannah, A., Luke, G., Wilson, K., Homan, K., Emelianov, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indocyanine+green-loaded+photoacoustic+nanodroplets:+dual+contrast+nanoconstructs+for+enhanced+photoacoustic+and+ultrasound+imaging.">Indocyanine green-loaded photoacoustic nanodroplets: dual contrast nanoconstructs for enhanced photoacoustic and ultrasound imaging.</a> ACS Nano. 8 (1), 250-259 (2013).</li><li>Kim, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-infrared+plasmonic+assemblies+of+gold+nanoparticles+with+multimodal+function+for+targeted+cancer+theragnosis.">Near-infrared plasmonic assemblies of gold nanoparticles with multimodal function for targeted cancer theragnosis.</a> Scientific Reports. 7 (1), 17327 (2017).</li><li>Ku, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Copper+sulfide+nanoparticles+as+a+new+class+of+photoacoustic+contrast+agent+for+deep+tissue+imaging+at+1064+nm.">Copper sulfide nanoparticles as a new class of photoacoustic contrast agent for deep tissue imaging at 1064 nm.</a> ACS Nano. 6 (8), 7489-7496 (2012).</li><li>Parker, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folate+receptor+expression+in+carcinomas+and+normal+tissues+determined+by+a+quantitative+radioligand+binding+assay.">Folate receptor expression in carcinomas and normal tissues determined by a quantitative radioligand binding assay.</a> Analytical Biochemistry. 338 (2), 284-293 (2005).</li><li>Cheung, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+folate+receptor+alpha+for+cancer+treatment.">Targeting folate receptor alpha for cancer treatment.</a> Oncotarget. 7 (32), 52553 (2016).</li><li>Zhou, M., Song, S., Zhao, J., Tian, M., Li, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theranostic+CuS+nanoparticles+targeting+folate+receptors+for+PET+image-guided+photothermal+therapy.">Theranostic CuS nanoparticles targeting folate receptors for PET image-guided photothermal therapy.</a> Journal of Materials Chemistry B. 3 (46), 8939-8948 (2015).</li><li>Lusk, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoacoustic+Flow+System+for+the+Detection+of+Ovarian+Circulating+Tumor+Cells+Utilizing+Copper+Sulfide+Nanoparticles.">Photoacoustic Flow System for the Detection of Ovarian Circulating Tumor Cells Utilizing Copper Sulfide Nanoparticles.</a> ACS Biomaterials Science &amp; Engineering. 5 (3), 1553-1560 (2019).</li><li>Lee, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predictive+value+of+circulating+tumor+cells+(CTCs)+captured+by+microfluidic+device+in+patients+with+epithelial+ovarian+cancer.">Predictive value of circulating tumor cells (CTCs) captured by microfluidic device in patients with epithelial ovarian cancer.</a> Gynecologic Oncology. 145 (2), 361-365 (2017).</li><li>Blassl, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiling+of+single+circulating+tumor+cells+in+ovarian+cancer-Establishment+of+a+multi-marker+gene+panel.">Gene expression profiling of single circulating tumor cells in ovarian cancer-Establishment of a multi-marker gene panel.</a> Molecular Oncology. 10 (7), 1030-1042 (2016).</li><li>Lu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+living+circulating+tumor+cells+in+patients+by+immunomagnetic+negative+enrichment+coupled+with+flow+cytometry.">Isolation and characterization of living circulating tumor cells in patients by immunomagnetic negative enrichment coupled with flow cytometry.</a> Cancer. 121 (17), 3036-3045 (2015).</li><li>Bhattacharyya, K., Goldschmidt, B. S., Viator, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+capture+of+breast+cancer+cells+with+photoacoustic+flow+cytometry.">Detection and capture of breast cancer cells with photoacoustic flow cytometry.</a> Journal of Biomedical Optics. 21 (8), 087007 (2016).</li><li>Zharov, V. P., Galanzha, E. I., Shashkov, E. V., Khlebtsov, N. G., Tuchin, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+photoacoustic+flow+cytometry+for+monitoring+of+circulating+single+cancer+cells+and+contrast+agents.">In vivo photoacoustic flow cytometry for monitoring of circulating single cancer cells and contrast agents.</a> Optics Letters. 31 (24), 3623-3625 (2006).</li><li>Galanzha, E. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+liquid+biopsy+using+Cytophone+platform+for+photoacoustic+detection+of+circulating+tumor+cells+in+patients+with+melanoma.">In vivo liquid biopsy using Cytophone platform for photoacoustic detection of circulating tumor cells in patients with melanoma.</a> Science Translational Medicine. 11 (496), eaat5857 (2019).</li><li>Cai, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+photoacoustic+flow+cytometry+for+early+malaria+diagnosis.">In vivo photoacoustic flow cytometry for early malaria diagnosis.</a> Cytometry Part A. 89 (6), 531-542 (2016).</li><li>Galanzha, E. 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This protocol is a straightforward method for the detection of ovarian CTCs utilizing PAFC and a targeted CuS contrast agent. Many methods have been explored for the detection of ovarian CTCs, including microfluidic devices, RT-PCR, and fluorescence flow cytometry23,24,25. These range in complexity, cost, and accuracy, limiting their effectiveness in clinical settings. PAFC introduces several advantages over these traditional me...

Ovarian cancer is one of the deadliest gynecological malignancies and resulted in an estimated 184,800 deaths worldwide in 20181. Multiple studies have shown the correlation between ovarian cancer progression (i.e., metastasis) and the presence of CTCs2,3,4. The most common method for detection and isolation of CTCs utilizes the Cellsearch system, which targets the EpCam receptor5. EpCam expression, however, is downregulated in epithelial to mesenchymal transition, which has been implicated in cancer metastasis6. Despite advances, current clinical technologies still suffer from low accuracy, high cost, and complexity. Due to these drawbacks, new technologies for the discovery and enumeration of ovarian CTCs has become an important area for research.
Recently, PAFC emerged as an effective method for the noninvasive detection of cancer cells, analysis of nanomaterials, and identification of bacteria7,8,9. PAFC differs from traditional fluorescence flow cytometry by detecting analytes in flow by utilizing photoacoustics. The photoacoustic effect is generated when laser light is absorbed by a material that causes thermoelastic expansion, producing an acoustic wave that can be detected by an ultrasound transducer10,11. Advantages of PAFC over traditional flow cytometry methods include simplicity, ease of translation to clinical settings, and the detection of CTCs at unprecedented depths in patient samples12,13. Recent studies have utilized PAFC systems for the detection of cells using endogenous and exogenous contrast14,15. Near infrared (NIR) light-absorbing contrast agents such as indocyanine green dye, and metal NPs (e.g., gold and CuS) have been used for the selective labeling of cells and tissues in combination with photoacoustic imaging16,17,18. Due to the improved penetration depth of NIR light within biological tissues, photoacoustic detection of absorbers can be performed at greater depths for clinical applications. Because of its great potential for use in the clinic, the combination of targeted NIR contrast agents with PAFC has generated considerable interest for the detection of CTCs.
PAFC in combination with targeted contrast agents provides an improved approach for high-throughput analysis of patient samples with enhanced accuracy and targeted detection of CTCs. One of the principal detection strategies for CTCs is the specific targeting of membrane proteins present on the cell of interest. One notable characteristic of ovarian CTCs is the overexpression of folate receptors located on their outer membrane19. Folate receptor targeting is an ideal strategy for the identification of ovarian CTCs in blood because endogenous cells, which have higher expression of folic acid receptors, are generally luminal and have limited exposure to the bloodstream20. Copper sulfide NPs (CuS NPs) have recently been recognized for their ability to target folate receptors expressed on cancerous cells21. Combined with their biocompatibility, ease of synthesis, and absorption deep in the NIR, these NP contrast agents make an ideal targeting strategy for the detection of ovarian CTCs utilizing PAFC.
This work describes the preparation of FA-CuS NPs and their use for the detection of ovarian cancer cells in a photoacoustic flow system. CuS NPs are modified with folic acid to specifically target ovarian CTCs and emit a photoacoustic signal when stimulated with a 1,053 nm laser. The results indicate the successful detection of ovarian cancer cells incubated with these photoacoustic contrast agents within the PAFC system. These results show detection of ovarian cancer cells down to concentrations of 1 cell/&#181;L, and fluorescence microscopy confirms successful uptake of these particles by SKOV-3 ovarian cancer cells22. This work provides a detailed description of the FA-CuS NPs synthesis, preparation of samples for fluorescence microscopy, construction of the photoacoustic flow system, and the photoacoustic detection of ovarian cancer cells. The presented method shows successful identification of ovarian CTCs in flow utilizing FA-CuS NPs. Future work will focus on the clinical application of this technology towards the early detection of ovarian cancer metastasis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.025% Trypsin With EDTA</td>
			<td>Corning</td>
			<td>25-053-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>0.2 &#181;m 1000 mL Vacuum Filtration Unit</td>
			<td>VWR</td>
			<td>10040-440</td>
			<td>For filtering larger volumes of DI water.</td>
		</tr>
		<tr>
			<td>0.2 &#181;m sterile syringe filter</td>
			<td>VWR</td>
			<td>28145-477</td>
			<td></td>
		</tr>
		<tr>
			<td>3D Printed Tank</td>
			<td></td>
			<td>Custom-made</td>
			<td></td>
		</tr>
		<tr>
			<td>Acquisition Card</td>
			<td>National Instruments</td>
			<td>PXIe-5170R</td>
			<td>250 MS/s, 8-Channel, 14-bit</td>
		</tr>
		<tr>
			<td>Alconox</td>
			<td>Sigma-Aldrich</td>
			<td>242985-1.8KG</td>
			<td>Detergent used for cleaning glassware.</td>
		</tr>
		<tr>
			<td>Amicon Ultra-15 Centrifugal Filters</td>
			<td>Millipore</td>
			<td>UFC903024</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-4 Centrifugal Filters</td>
			<td>Millipore</td>
			<td>UFC803024</td>
			<td></td>
		</tr>
		<tr>
			<td>Bright-Line Hematocytometer</td>
			<td>Hausser Scientific</td>
			<td>1492</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper(II) Chloride</td>
			<td>ACROS ORGANICS</td>
			<td>206532500</td>
			<td></td>
		</tr>
		<tr>
			<td>Coupling Objective</td>
			<td>Thorlabs</td>
			<td>LMH-10x-532</td>
			<td>To couple pulsed light to optical fiber.</td>
		</tr>
		<tr>
			<td>Coupling Stage</td>
			<td>Newport</td>
			<td>F-91-C1-T</td>
			<td>Stage for coupling pulsed light to objective. Holds FP-1A and LMH-10x-532</td>
		</tr>
		<tr>
			<td>CPX Series Digital Ultrasonic Cleaning Bath</td>
			<td>Fisherbrand</td>
			<td>Model CPX3800</td>
			<td></td>
		</tr>
		<tr>
			<td>Data Acquisition software</td>
			<td>National Instruments</td>
			<td>NI LabVIEW 2017 (32-bit)</td>
			<td>LabVIEW used to synchronize laser pulses with data acquisition.</td>
		</tr>
		<tr>
			<td>Data Processing Software</td>
			<td>Mathworks</td>
			<td>Matlab R2016a</td>
			<td>Reconstructions and graphs produced using Matlab software.</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F2442-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber Chuck</td>
			<td>Newport</td>
			<td>FPH-DJ</td>
			<td>Used to hold the bare fiber.</td>
		</tr>
		<tr>
			<td>Fiber Coupler</td>
			<td>Newport</td>
			<td>FP-1A</td>
			<td>3-Axis stage for positioning fiber chuck and optical fiber at the focus of the objective.</td>
		</tr>
		<tr>
			<td>Folic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>F7876-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Formvar Coated TEM Grids</td>
			<td>Electron Microscopy Sciences</td>
			<td>FCF300-CU-SB</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex Tubing</td>
			<td>Cole Parmer</td>
			<td>EW-96420-14</td>
			<td></td>
		</tr>
		<tr>
			<td>McCoy&#39;s 5A Medium</td>
			<td>ATCC</td>
			<td>30-2007</td>
			<td></td>
		</tr>
		<tr>
			<td>Norm-Ject 10 mL Syringes</td>
			<td>HENKE SASS WOLF</td>
			<td>4100-X00V0</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical Fiber</td>
			<td>Thorlabs</td>
			<td>FG550LEC</td>
			<td>Used to expose sample to pulsed light.</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Alfa Aesar</td>
			<td>J62036</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>GIBCO</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Pulsed Laser</td>
			<td>RPMC Lasers Inc</td>
			<td>Quantus-Q1D-1053</td>
			<td>Pulsed laser source with specifications 1053 nm, 8 ns pulse, 10 Hz maximum.</td>
		</tr>
		<tr>
			<td>Pulser/Receiver</td>
			<td>Olympus</td>
			<td>5077PR</td>
			<td>Receives, filters, and amplifies photoacoustic signals. Operated with 59 dB Gain.</td>
		</tr>
		<tr>
			<td>Quartz Capillary Tube</td>
			<td>Sutter Instrument</td>
			<td>QF150-75-10</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI Midum 1640 (1X) Folic Acid Free</td>
			<td>Gibco</td>
			<td>27016-021</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone</td>
			<td>Momentive Performance Materials, Inc.</td>
			<td>GE284</td>
			<td></td>
		</tr>
		<tr>
			<td>SKOV-3 Cells</td>
			<td>ATCC</td>
			<td>HTB-77</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S7795-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide Beads</td>
			<td>BDH</td>
			<td>BDH9292-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Sulfide Nonahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>431648-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Pumps</td>
			<td>New Era Pump Systems Inc</td>
			<td>DUAL-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Texas Red-X-Succinimydl ester</td>
			<td>Invitrogen</td>
			<td>1949071</td>
			<td></td>
		</tr>
		<tr>
			<td>Transducer</td>
			<td>Olynmpus</td>
			<td>V214-BB-RM</td>
			<td>Ultrasound detector with central frequency of 50 MHz and -6 dB fractional bandwidth of 82%.</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution .4%</td>
			<td>Amresco</td>
			<td>K940-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P7949-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound Gel</td>
			<td>Parker Laboratories Inc.</td>
			<td>Aquasonic 100</td>
			<td>Ultrasound gel for transducer coupling</td>
		</tr>
	</tbody>
</table>

ovarian cancer detection using photoacoustic flow cytometry

Many studies suggest that the enumeration of circulating tumor cells (CTCs) may show promise as a prognostic tool for ovarian cancer. Current strategies for the detection of CTCs include flow cytometry, microfluidic devices, and real-time polymerase chain reaction (RT-PCR). Despite recent advances, methods for the detection of early ovarian cancer metastasis still lack the sensitivity and specificity required for clinical translation. Here, a novel method is presented for the detection of ovarian circulating tumor cells by photoacoustic flow cytometry (PAFC) utilizing a custom three dimensional (3D) printed system, including a flow chamber and syringe pump. This method utilizes folic acid-capped copper sulfide nanoparticles (FA-CuS NPs) to target SKOV-3 ovarian cancer cells by PAFC. This work demonstrates the affinity of these contrast agents for ovarian cancer cells. The results show NP characterization, PAFC detection, and NP uptake by fluorescence microscopy, thus demonstrating the potential of this novel system to detect ovarian CTCs at physiologically relevant concentrations.

Figure 1A shows a typical TEM image of the synthesized nanoparticles. The average size of the typical nanoparticle is approximately 8.6 nm &#177; 2.5 nm. Nanoparticle measuring was performed in ImageJ. Threshold and watershed functions were applied to separate the particles for measurement. The horizontal and vertical diameters of each particle were measured perpendicular to each other and further averaged. For DLS, a representative measureme...

Watch this Scientific Journal Video about Ovarian Cancer Detection Using Photoacoustic Flow Cytometry at JoVE.com

Ovarian Cancer Detection Using Photoacoustic Flow Cytometry

A protocol is presented to detect circulating ovarian tumor cells utilizing a custom-made photoacoustic flow system and targeted folic acid-capped copper sulfide nanoparticles.

Many studies suggest that the enumeration of circulating tumor cells (CTCs) may show promise as a prognostic tool for ovarian cancer. Current strategies ...

Current clinical ovarian cancer detection measures are nonspecific and lack point of care detection of circulating tumor cells. Our photoacoustic flow system enables testing patient samples for specific ovarian cancer markers in the blood stream. This procedure provides a unique platform technology with improvements over the current techniques in terms of simplicity, low cost, promising limits of detection, and ability to be applied to broad applications.Through this work, we aim to build off of our targeted nanoparticle system for ovarian cancer to test ex vivo patient samples for CTCs. This highly versatile PAFC system is able to expand for clinical use, testing the presence of a wide range of biomarkers in the blood. Proper optical alignment is critical for detection of targets within the photoacoustic flow cytometry system.Furthermore, to ensure proper acoustic coupling, make sure there are no bubbles between the glass slide and transducer. Because of the custom nature of the flow system and its components, visualization of the method is beneficial for accurate reproduction. In a ventilated chemical fume hood, filter approximately 300 milliliters of deionized water through a sterile 0.2 micrometer filter.Add 0.0134 grams of copper chloride and 100 milliliters of deionized water to a clean 250 milliliter round bottom flask to create a one millimolar copper chloride solution. Add 0.015 grams of folic acid to the flask and use a magnetic stir bar to mix the solution for approximately five minutes. Then mix 0.024 grams of sodium sulfide nonahydrate with 100 microliters of deionized water.Use a 200 microliter pipette to slowly add this solution to the reaction mixture over a duration of approximately 10 seconds. Cap the reaction vessel. Place the vessel in an oil bath set to 90 degrees Celsius and continue stirring.After approximately 15 minutes or when the oil bath has reached the temperature range between 85 and 90 degrees Celsius, allow the reaction to proceed for an additional hour. When the reaction is complete, remove the reaction vessel from the oil bath and let it cool at room temperature for 10 to 15 minutes before transferring it to an ice bath. Once the reaction is cooled, adjust the pH to approximately 10 using one molar sodium hydroxide.Add the mixture to a 30 kilodalton centrifugation column in 15 milliliter batches and centrifuge at 3, 082 times g for 15 minutes to purify the reaction mixture. First, mix the folic acid capped copper sulfide nanoparticles at the proper concentration in fresh RPMI media. Add this nanoparticle solution to each well of the prepared 24-well plate that contains SK-OV-3 cells at a concentration of 400 micrograms per milliliter.Incubate at 37 degrees Celsius with 5%carbon dioxide for two hours. After this, trypsinize the cells with 0.5 milliliters of 0.25%trypsin and EDTA. To neutralize the trypsin, add at least one milliliter of folic acid-free RPMI 1640 complete growth media and centrifuge the cells at 123 times g for six minutes.To wash the cells, remove the supernatant, resuspend the cells in two milliliters of PBS and centrifuge at 123 times g for six minutes. Repeat this wash step twice to remove any unbound nanoparticles. Then resuspend the cells with one to two milliliters of a 2%Tween solution in PBS.Count the cells using a hemocytometer and Trypan Blue. Dilute the cells in a solution of 2%Tween in PBS to the chosen concentration for detection. Use the provided three-dimensional STL file to 3D print the flow tank with either ABS thermoplastic or PLA plastic.After printing the tank, clean and assemble the system for use. Place glass coverslips over the one millimeter by three millimeter slot and the one centimeter hole in the flow system and carefully seal with silicone to prevent leakage. Next, fit the capillary tube into the silicone-cured tubes.Insert the tubes into the flow chamber through the side of the flow tank such that the glass capillary tube is directly above and in front of the three millimeter slot and the one centimeter hole. Seal the tubing with silicone. Then connect the transducer to an ultrasound pulser and receiver.Amplify the signal with a 59 decibel gain. Connect the output of the filter to a multipurpose reconfigurable oscilloscope equipped with a built-in field programmable gate array. Connect one of the tubes coming from the flow chamber to a T junction that is connected to two syringe pumps at each branch.Fill one of the syringe pumps with air and the other pump with the sample to be analyzed. Set the pump containing air to a flow rate of 40 microliters per minute and set the pump containing the sample to a flow rate of 20 microliters per minute. Next, connect the remaining tube exiting the flow system to a container of 10%bleach to dispose of cells after they exit the flow system.Place the section of the quartz capillary tube in direct alignment with the transducer in the field view of the microscope to allow for careful placement of the optical fiber above the sample such that it illuminates the entire width of the tube. Irradiate the sample using an optical fiber channeling a diode-pumped solid-state laser operating at a wavelength of 1, 053 nanometers. Use a microscope mounted camera to record the flow, the firing of the laser, and the passage of samples through the flow system.In this study, photoacoustic flow cytometry and the targeted copper sulfide targeting agent are used to detect ovarian circulating tumor cells. A typical TEM image of the synthesized nanoparticles show that the average size of a typical nanoparticle is approximately 8.6 nanometers. The horizontal and vertical diameters of each particle are then measured perpendicular to each other and further averaged.The average hydrodynamic diameter for these particles is 73.6 nanometers. Copper sulfide nanoparticles have a characteristic absorbance curve which extends into the near infrared. There is a slight artifact around 850 nanometers that is caused by the switching of lasers by the spectrophotometer.Fluorescence microscopy images of cells incubated with fluorescently tagged nanoparticles show that nanoparticle uptake can be visualized by the presence of fluorescence across the cell. Cells not incubated with nanoparticles show no fluorescent signal. The presence of this fluorescent signal indicates the successful uptake of the particles and their ability to be detected in the flow system.Examples of typical data acquisition signals are shown here. The raw data indicates the differences in signal between the nanoparticle tagged cells, PBS, and folic acid capped copper sulfide nanoparticles. Utilizing custom lab view and MATLAB software, image reconstructions are made of the positive and negative controls in real time and post acquisition respectively.Individual envelopers are subsequently converted into pixel values and displayed as independent columns. Clear differences in photoacoustic signal occur between PBS and the folic acid capped copper sulfide nanoparticles at a concentration of 100 micrograms per milliliter. During this procedure, it is critical to properly align the ultrasound transducer, microscope and fiber optic within the photoacoustic flow cytometry system.Confirm system alignment by testing positive and negative controls. Due to the versatility of photoacoustic targeting techniques and the wide range of applications possible using PAFC, this technique paves the way for translationally important clinical and research applications. To ensure clinical application, further studies should focus on the testing of patient samples and the reduction of procedural steps.The synthesis of the nanoparticles should take place in a chemical fume hood utilizing the proper protective equipment. Human cell lines must be handled according to your institution's guidelines. The use of the laser requires radiation safety training and appropriate PPE.Laser usage and photoacoustic testing should only be performed by highly trained personnel.

ovarian-cancer-detection-using-photoacoustic-flow-cytometry

Research

JoVE Journal

Bioengineering

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors1,2,3. CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient&prime;s response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)4,5. This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid6,7. PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.

We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

Photoacoustic Cystography

Conventional pediatric cystography, which is based on diagnostic X-ray using a radio-opaque dye, suffers from the use of harmful ionizing radiation. The risk of bladder cancers in children due to radiation exposure is more significant than many other cancers. Here we demonstrate the feasibility of nonionizing and noninvasive photoacoustic (PA) imaging of urinary bladders, referred to as photoacoustic cystography (PAC), using near-infrared (NIR) optical absorbents (i.e. methylene blue, plasmonic gold nanostructures, or single walled carbon nanotubes) as an optical-turbid tracer. We have successfully imaged a rat bladder filled with the optical absorbing agents using a dark-field confocal PAC system. After transurethral injection of the contrast agents, the rat's bladders were photoacoustically visualized by achieving significant PA signal enhancement. The accumulation was validated by spectroscopic PA imaging. Further, by using only a laser pulse energy of less than 1 mJ/cm2 (1/20 of the safety limit), our current imaging system could map the methylene-blue-filled-rat-bladder at the depth of beyond 1 cm in biological tissues in vivo. Both in vivo and ex vivo PA imaging results validate that the contrast agents were naturally excreted via urination. Thus, there is no concern regarding long-term toxic agent accumulation, which will facilitate clinical translation.

Photoacoustic cystography (PAC) has a great potential to map urinary bladders, a radiation sensitive internal organ in pediatric patients, without using any ionizing radiation or toxic contrast agent. Here we demonstrate the use of PAC for mapping urinary bladders with an injection of optical-opaque tracers in rats in vivo.

Conventional pediatric cystography, which is based on diagnostic X-ray using a radio-opaque dye, suffers from the use of harmful ionizing radiation. The ...

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. ~ 10 &mu;m over a very large field-of-view of ~ 81 mm2. This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water.

We review our recent results on the integration of fluorescent microscopy and imaging flow cytometry tools on a cell-phone using compact and cost-effective opto-fluidic attachments. These cell-phone based micro-analysis devices might be useful for cytometric analysis, such as performing various cell counting tasks as well as for high-throughput screening of e.g., water samples in resource limited settings.

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general ...

Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and progression of pathology in several neurodegenerative diseases, including Alzheimer's disease and the related tauopathies. The potentially critical role of tau seeds in disease progression strongly supports the need for a sensitive assay that readily detects seeding activity in biological samples. 
By combining the specificity of fluorescence resonance energy transfer (FRET), the sensitivity of flow cytometry, and the stability of a monoclonal cell line, an ultra-sensitive seeding assay has been engineered and is compatible with seed detection from recombinant or biological samples, including human and mouse brain homogenates. The assay employs monoclonal HEK 293T cells that stably express the aggregation-prone repeat domain (RD) of tau harboring the disease-associated P301S mutation fused to either CFP or YFP, which produce a FRET signal upon protein aggregation. The uptake of proteopathic tau seeds (but not other proteins) into the biosensor cells stimulates aggregation of RD-CFP and RD-YFP, and flow cytometry sensitively and quantitatively monitors this aggregation-induced FRET. The assay detects femtomolar concentrations (monomer equivalent) of recombinant tau seeds, has a dynamic range spanning three orders of magnitude, and is compatible with brain homogenates from tauopathy transgenic mice and human tauopathy subjects. With slight modifications, the assay can also detect seeding activity of other proteopathic seeds, such as &#945;-synuclein, and is also compatible with primary neuronal cultures. The ease, sensitivity, and broad applicability of FRET flow cytometry makes it useful to study a wide range of protein aggregation disorders.

Cell-to-cell transfer of protein aggregates, or proteopathic seeds, may underlie the progression of pathology in neurodegenerative diseases. Here, a novel FRET flow cytometry assay is described that enables specific and sensitive detection of seeding activity from recombinant or biological samples.

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and ...

Modeling Ovarian Cancer Multicellular Spheroid Behavior in a Dynamic 3D Peritoneal Microdevice

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with poor clinical outcomes and currently lacks effective treatment. Both the three-dimensional (3D) environment and the dynamic mechanical forces are very important factors in this metastatic cascade. However, traditional cell cultures fail to recapitulate this natural tumor microenvironment. Thus, in vivo-like models that can emulate the intraperitoneal environment are of obvious importance. In this study, a new microfluidic platform of the peritoneum was set up to mimic the situation of ovarian cancer spheroids in the peritoneal cavity during metastasis. Ovarian cancer spheroids generated under a non-adherent condition were cultured in microfluidic channels coated with peritoneal mesothelial cells subjected to physiologically relevant shear stress. In summary, this dynamic 3D ovarian cancer-mesothelium microfluidic platform can provide new knowledge on basic cancer biology and serve as a platform for potential drug screening and development.

To study ovarian tumor progression in a physiologically relevant model, multicellular spheroids were cultured in a microdevice under simulated fluid flow. This dynamic 3D model emulates the intraperitoneal environment with the cellular and mechanical components where ovarian cancer metastasis occurs.

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with ...

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...