I would like to introduce the novel methods to efficiently detect and harvest circulating tumor cells in the bloodstream.

Prior to running blood through the CTC‐iChip, blood was analyzed by a cell blood count machine to determine total white blood cell (WBC) count. Biotinylated primary antibodies against anti‐human CD45 antibody (clone no. 2D1, BAM1430; R&D Systems, Minneapolis, MN, http://www.rndsystems.com) and anti‐human CD66b antibody (80H3; Bio‐Rad Laboratories [formerly AbD Serotec], Hercules, CA, http://www.bio-rad-antibodies.com) were spiked into whole blood at 100 femtograms per WBC (fg/WBC) and 37.5 fg/WBC, respectively, and incubated rocking at room temperature for 20 minutes. Dynabeads MyOne Strepavidin T1 (65602; Thermo Fisher Scientific, Waltham, MA, http://thermofisher.com) magnetic beads were then added and incubated, rocking at room temperature, for an additional 20 minutes.

After whole‐blood CTC‐iChip processing, the product containing enriched cells was fixed with 4% paraformaldehyde and an aliquot was stained with fluorescently labeled antibodies targeting epithelial antigens (CK8/18‐AF488, CK19‐AF488, EpCAM‐AF555; Janssen, Beerse, Belgium, http://www.janssen.com), leukocyte antigens (CD16‐AF647, CD45‐AF647, CD66b‐AF647; BioLegend, San Diego, CA, http://www.biolegend.com), and DAPI (5 μg/mL; Thermo Fisher). Slides were mounted using ProLong Gold Antifade Reagent (Thermo Fisher). Fixed and stained cells were imaged by fluorescence microscopy (TiE or Eclipse 90i; Nikon Instruments, Melville, NY, http://www.nikoninstruments.com) using the appropriate filter cubes for image acquisition and the BioView platform (BioView, Billerica, MA, http://bioview.com) for automated image analysis. All candidate CTCs detected were reviewed and scored based on intact morphology, colocalization of CTC markers with DAPI nuclear counterstain, and absence of leukocyte markers.

A second aliquot was processed for RNA in situ hybridization (RNA‐ISH) using fluorescent probes directed against HSATII and keratin. ISH was performed according to the Affymetrix ViewRNA ISH Cell Assay (Thermo Fisher). Briefly, cells were fixed in 4% formaldehyde for 30 minutes at room temperature, permeabilized by pretreating in buffer solution for 5 minutes at room temperature, and digested with protease for 10 minutes. Target probe sets were applied and hybridized to the cells by incubating for 3 hours at 40°C. Probes were used at a dilution of 1:50 for Type 1 HSATII and 1:50 for Type 6 keratin 7, 8, 18, and 19 (Affymetrix; Thermo Fisher). Signal was amplified through the sequential hybridization of PreAmplifier and Amplifier mixes to the target probe set, and target RNA molecules were detected by applying Label Probe Mix (Thermo Fisher). Cells were then counterstained with DAPI (5 μg/ml; Thermo Fisher) and slides were mounted using ProLong Gold Antifade Reagent (Thermo Fisher). Fluorescence microscopy using a Nikon 90i was used to visualize target mRNA transcripts. Type 1 probes were detected in the Cy3 channel and Type 6 probes in the Cy5 channel. Merged images were generated using NISElements software (Nikon). Colocalization analysis of HSATII and DAPI was performed using Zen software on 10 single cells (×63 magnification) imaged by LSM 710 Confocal Microscope (ZEISS, Oberkochen, Germany, http://www.zeiss.com). A Manders overlap coefficient R ≥ 0.6 indicates signal colocalization. In some patients, due to technical (sample processing) issues, either IF or ISH data were unable to be collected. For the CEC data analysis, only the data from those patients for whom both immunofluorescence and ISH were satisfactorily performed were used in our summary statistics. Counts were normalized as CECs per mL of whole blood.

A third aliquot was pelleted and flash frozen in RNAlater (Thermo Fisher) at −80°C. RNA was extracted (RNEasy Micro; Qiagen, Hilden, Germany, http://www.qiagen.com) and processed as follows for RNA‐seq. Amplified cDNA was generated from RNA from each sample using the SMARTer Ultra Low Input RNA Kit for Sequencing (version 3 or 4; Clontech Laboratories, Mountain View, CA, http://www.clontech.com) according to the manufacturer's protocol. Briefly, 1 μL of a 1:50,000 dilution of ERCC RNA Spike‐In Mix (Thermo Fisher [formerly Life Technologies]) was added to each sample. First‐strand synthesis of RNA molecules was performed using the poly‐dT‐based 3’‐SMART CDS primer II A (Clontech) followed by extension and template switching by the reverse transcriptase. The second strand synthesis and amplification polymerase chain reaction (PCR) was run for 18 cycles, and the amplified cDNA was purified with a 1× Agencourt AMPure XP bead cleanup (Beckman Coulter, Brea, CA, http://www.beckmancoulter.com). The Nextera XT DNA Library Preparation kit (Illumina, San Diego, CA, http://www.illumina.com) was used for sample barcoding and fragmentation according to the manufacturer's protocol. One nanogram of amplified cDNA was used for the enzymatic tagmentation, followed by 12 cycles of amplification and unique dual‐index barcoding of individual libraries. PCR product was purified with a 1.8× Agencourt AMPure XP bead cleanup (Beckman Coulter). The eluted cDNA libraries did not undergo the bead‐based library normalization step in the Nextera XT protocol. Library validation and quantification was performed by quantitative PCR using the KAPA SYBR FAST Universal quantitative polymerase chain reaction (qPCR) Kit (Kapa Biosystems, Wilmington, MA, http://www.kapabiosystems.com). The individual libraries were pooled at equal concentrations, and the pool concentration was determined using the KAPA SYBR FAST Universal qPCR Kit. The pool of libraries was subsequently sequenced in three replicates on a HiSeq 2500 (Illumina) in Rapid Run Mode using a 2 × 100 base pair kit and a dual flow cell. The paired‐end reads from the three sequencing runs were combined and aligned to the hg38 genome from the University of California, Santa Cruz (http://genome.ucsc.edu) using the STAR version 2.4.0h aligner with default settings (Alex Dobin, https://github.com/alexdobin/STAR). Reads that did not map or mapped to multiple locations were discarded. Duplicate reads were marked using the MarkDuplicates tool in picard‐tools‐1.8.4 (Broad Institute, broadinstitute.github.io/picard) and were removed. The uniquely aligned reads were counted using htseq‐count in the intersection‐strict mode against the Homo_sapiens.GRCh38.79.gtf annotation table from Ensembl (Hinxton, Cambridge, United Kingdom, www.ensembl.org). Data were then imported into the R statistical programming language for analysis and heat map generation (median‐normalized log 10‐tranformed reads per million plus 1 to allow for log transformation). All RNA‐seq raw data has been submitted to National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO).

【Reference】

Improved Detection of Circulating Epithelial Cells in Patients with Intraductal Papillary Mucinous Neoplasms

The Oncologist 2018; 23:121-127; first published on August 31, 2017;doi:10.1634/theoncologist.2017-0234

Hello

I suggest this research group of Turkey

Haluk Kulah

http://mikrobiyo.com.tr/

For any other types of microfluidic application we will be glad to help you with our free demo service.

Best wishes

Serhat

www.nehirbt.com.tr

What we do is process the blood with Ficoll then FACS sort the cell population and look for the CD45 negative population on FACS

Hello

kindly look at these links :

Article Selective isolation and noninvasive analysis of circulating ...

Article Circulating cancer stem cells: The importance to select

Article Methods for Detecting Circulating Cancer Stem Cells (CCSCs) ...

Good Luck