CTCs are isolated according to their physical properties by Ficoll density gradient centrifugation. In short, whole blood is layered on Ficoll and then centrifuged for 30 min at room temperature. Ery-lysis is done for 3 min and isolated peripheral mononuclear blood cells are cyto-centrifuged onto glass slides (500000 cells per slide)35 (link). For detection of CTCs, immunofluorescence stainings were performed using the pan-cytokeratin antibody (1:80; AE1AE3, eBioScience) and ALDH1 antibody (1:100; ab52492, Abcam) in combination with a species-specific secondary antibody (1:200; anti-rabbit-Alexa488, LifeTechnologies), and a CD45 antibody (1:150; Clone HI30, BioLegend). Cells were fixed with 0.5% para-formaldehyde and blocked with AB serum. All antibodies were incubated for 45 min at room temperature except for ALDH1 (overnight, 4 °C). Glass slides were covered and manually analysed by fluorescence microscopy.
Clone hi30
Manufactured by BioLegend
Sourced in United States
The Clone HI30 is a laboratory equipment product. It is used for the detection and analysis of specific cell surface markers.
Automatically generated - may contain errors
Lab products found in correlation
Cell id cisplatin, by Standard BioTools (2 mentions)
Maxpar cell staining buffer, by Standard BioTools (2 mentions)
Human trustain fcx, by BioLegend (2 mentions)
Falcon 5 ml 12 75 mm tubes, by Corning (2 mentions)
89y cd45, by Standard BioTools (2 mentions)
Ab52492, by Abcam (1 mentions)
Alexa 488 anti rabbit, by Thermo Fisher Scientific (1 mentions)
Ae1 ae3, by Thermo Fisher Scientific (1 mentions)
Facs tube, by BD (1 mentions)
Clone hit3a, by BioLegend (1 mentions)
Rpa t8, by BioLegend (1 mentions)
Clone rpa t4, by BioLegend (1 mentions)
Nod cg prkdcscid il2rgtm1wjl szj nsg mice, by Jackson ImmunoResearch (1 mentions)
Nsg mice, by Jackson ImmunoResearch (1 mentions)
Clone p67, by BioLegend (1 mentions)
Zombie live dead stain, by BioLegend (1 mentions)
Clone ucht1, by BioLegend (1 mentions)
Clone hib19, by BioLegend (1 mentions)
Facsdiva, by BD (1 mentions)
Streptavidin af488, by Thermo Fisher Scientific (1 mentions)
12 protocols using clone hi30
1
CTC Isolation and Characterization by Immunofluorescence
2
Multiplexed Cell Barcoding Protocol
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CD45 antibodies (Cat No: 304002; Clone HI30; Biolegend, CA) were labeled with Tb159, Ho165, Tm169, and Lu175 (DVS Science, CA) as described; 5 ng of barcoding antibodies were added to the desired wells containing 1–3 million cells and incubated for 20 min. Following a wash with staining buffer (2% BSA in PBS), all barcoded samples were pooled into a single FACS tube (BD Sciences, NJ). This was followed by the cell staining procedure, which employs the protocols provided by DVS Science, including cisplatin (Cat No: 201064; DVS Science, CA) for live/dead cell staining, surface biomarker cocktail staining, intracellular marker staining and DNA intercalator-Ir (Cat No: 201192; DVS Science, CA) staining.
m-DOTA (Cat No: B-272-100; Macrocyclics, Dallas, TX) based barcode was prepared following the protocol described by Zivanovic et al. 9 (link). In summary, m-DOTA barcodes were prepared at 2:1 molar ratio of m-DOTA/metal in 20 mM acetate buffer, pH 5.5, lyophilized and resuspended in DMSO as a 10 mM stock. For barcoding, a final concentration of 250 nM to 1 µM was adopted based on the titration results (Fig. S2). After cisplatin staining, cells were fixed and permeabilized before the barcode reagents were added. The cells were transferred into one single tube, followed by full panel antibodies cocktail staining and DNA intercalator staining.
m-DOTA (Cat No: B-272-100; Macrocyclics, Dallas, TX) based barcode was prepared following the protocol described by Zivanovic et al. 9 (link). In summary, m-DOTA barcodes were prepared at 2:1 molar ratio of m-DOTA/metal in 20 mM acetate buffer, pH 5.5, lyophilized and resuspended in DMSO as a 10 mM stock. For barcoding, a final concentration of 250 nM to 1 µM was adopted based on the titration results (Fig. S2). After cisplatin staining, cells were fixed and permeabilized before the barcode reagents were added. The cells were transferred into one single tube, followed by full panel antibodies cocktail staining and DNA intercalator staining.
3
PBMC Viability Staining and Immunophenotyping
4
Generating Humanized Mice Models
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Hu-PBL and Hu-HSC hu-mice were generated from the xenoengraftment of human hematopoietic cells into immunocompromised NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice (Jackson Laboratories, Bar Harbor, ME), maintained under the daily care of the Yale Animal Resources Center (YARC). For generating Hu-HSC mice, 1-2-day old neonatal NSG mice (Jackson Laboratories, Bar Harbor, Maine) were irradiated at 100 cGy and injected through the intracardiac route with 1 x 105 CD34+ hematopoietic stem cells purified from human umbilical cord blood (National Disease Research Interchange). Hu-PBL mice were generated by i.p. injection of 1 x 107 PBMCs stored on ice into 2-4-month-old NSG mice. Mice were analyzed via flow cytometry after 10 weeks for Hu-HSC or after 2 weeks for Hu-PBL for the successful engraftment of monoclonal mouse anti-human CD45 (BioLegend, clone HI30), monoclonal mouse anti-human CD3 (BioLegend, clone HIT3a), monoclonal mouse anti-human CD4 (BioLegend, clone RPA-T4), and monocloncal mouse anti-human CD8 (BioLegend, clone RPA-T8) positive cells until all mice exhibited greater than 25% human CD45 expressing cells.
5
Characterizing Human Cell Engraftment in Humanized Mice
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For the analysis of human cell engraftment in humanized mice, recipient splenocytes and bone marrow cells were stained with Zombie live/dead stain (cat.no. 77184, Biolegend), anti-human CD45 (clone HI30, cat.no. 304008, 1:20 dilution, Biolegend with IgG1, PE, clone MOPC-21, cat.no. 400140 1:20 dilution, Biolegend), anti-human CD3 (clone UCHT1, cat.no. 300415, 1:20 dilution, Biolegend with IgG1, AF488, clone MOPC-21, cat.no. 400129 1:20 dilution, Biolegend), anti-human CD19 (clone HIB19, cat.no. 302228, 1:20 dilution, Biolegend with IgG1, PerCP, clone MOPC-21, cat.no. 400148 1:20 dilution, Biolegend) and anti-human CD33 (clone P67.6, cat.no. 366612, 1:20 dilution, Biolegend with IgG1, BV605, clone MOPC-21, cat.no. 400162 1:20 dilution, Biolegend). Cells were measured by flow cytometry (BD Aria and FACSDiva version 9.0 or CytExpert version 2.4 software) and gated for live cells, CD45 + cells and then followed by CD3 + cells, CD19 + or CD33 + cells. Results were expressed as percentage of positive population compared to control samples in FlowJo (BD).
6
Detecting Circulating Tumor Cells in NSCLC
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Thirty-seven metastatic NSCLC patients were screened for CTCs using a microfluidic device (Parsortix, Angle).25 (link) Collected in EDTA tubes was 7.5 mL of peripheral blood, which was processed immediately by the Parsortix device, followed by a cytocentrifugation of the obtained cells onto slides. The cells were visualized by immunofluorescence staining using antibodies against human keratins (1:100, clone AE1/AE3, eBioscience), ALCAM (1:150, clone 3A6, Biolegend), and CD45 (1:150; clone HI30, Biolegend). CTCs were defined as keratin+/4′,6′-diamidino-2-phenylindole positive (DAPI+)/CD45−. See also Supplementary Material.
7
Immunophenotyping of Hematopoietic Subsets
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Frozen BM aspirates were thawed on ice and stained with biotinylated antibodies against CD45 (1:50, clone HI30; BioLegend) and CD235a (1:50, clone HIR2, BioLegend) followed by depletion using magnetic anti-biotin beads (20 μL per 107 cells; Miltenyi Biotec) in PBS supplemented with 2% FCS and iMag (BD Biosciences). After depletion, the remaining cells were stained in PBS containing 0.5% FCS at 4°C with the following antibodies: Streptavidin-AF488 (1:100, Invitrogen), CD45-BV510(1:50, clone HI30; BioLegend), CD235a-PE-Cy7(1:50, clone HI264; BioLegend), CD71-AF700 (1:20, clone MEM-75; Exbio), CD271-PE (1:50, clone ME20.4; BioLegend), CD31-APC-Cy7 (1:20, clone WM59; BioLegend), and CD44-APC (1:50, clone IM7, Sony). 7AAD (1:100; Beckman Coulter) was used for dead cell exclusion. Samples were measured using FACSymphony A5 Cell Analyzer and analyzed with a FlowJo_v10.6.1 program.
8
Contact-dependent NK Cytotoxicity Assay
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Contact-dependence of pNK cytotoxicity against dl922-947-infected TOV21G was evaluated by assessing cell death of dl922-947-infected TOV21G in 0.4 μm transwells. TOV21G (104/well) were plated on the bottom of a 12-well plate and on the 0.4-μm-pore polycarbonate 12-well transwell inserts (12 mm membrane diameter; cat number: 3401, Corning, USA). After mock or dl922-947 infection (MOI 10, 48 h), pNK (105; ET 10:1), or no-NK culture medium (negative control) was added to the bottom of the well. After an 18-h incubation, the inserts were carefully removed without spillage into a separate 12-well plate. The supernatants and trypsinized TOV21G of each group were combined and centrifuged (430 g for 5 min) before staining with viability dye and CD45 antibody (Clone HI30; Biolegend, San Diego, CA). Cell death in CD45-negative target TOV21G cells was determined by flow cytometry.
9
PBMC Viability Staining and Immunophenotyping
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Washed PBMCs were resuspended in an appropriate volume of PBS to obtain a cell concentration of 107 cells/mL. Cells were incubated with a viability reagent, Cell-ID Cisplatin (Fluidigm, South San Francisco, CA) at a final concentration of 5 μM for 5 min on ice. Cisplatin was quenched by washing once with 5x volume of MaxPar® Cell Staining Buffer (Fluidigm, South San Francisco, CA) and centrifuged at 300 × g, then resuspended to a final concentration of 30 million cells/mL in staining buffer. To start antibody labeling, 3 million cells were transferred to Falcon® 5 mL 12 × 75 mm tubes (Corning, Corning, NY) and incubated with 5 μL of Human TruStain FcX (BioLegend, San Diego, CA) for 10 min on ice to block Fc receptor binding. Healthy PBMCs were stained with CD45-198Pt (inhouse conjugation of Clone HI30, Biolegend, San Diego, CA) as a reference to incorporate into each tumor sample as our internal reference control and tumor cells were stained with CD45-89Y (Fluidigm, South San Francisco, CA) for 30 min on ice. Cells were washed twice with 4 mL cell staining buffer before being prepared for surface staining.
10
Single-Cell Isolation and Profiling of Human Skin Cells
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Single-cell suspensions from human skin samples of adult subjects obtained as described in the section “Isolation of adult primary skin LECs and BECs from biopsies” were prepared as described above. Subsequently, isolated single cells were stained with mouse anti-human CD34 biotinylated antibody (clone 581, Thermo Fisher Scientific) diluted 5 µL for 106 cells in FACS buffer (DPBS with 2% FBS and 1 mM EDTA) for 30 min at 4 °C. After washing once with FACS buffer, isolated single cells were co-stained in FACS buffer with FITC-conjugated mouse anti-human CD45 antibody (1:25; clone HI30, Biolegend), PE-conjugated mouse anti-human CD31 antibody (1:25; clone WM59, BD Pharmingen), PerCP-conjugated streptavidin (1:400; Biolegend), Zombie NIR (1:500; BioLegend) for 30 min at 4 °C. After washing in FACS buffer, isolated single cells were filtered and sorted for living, CD45-CD31 + CD34low (LEC), or CD34 high (BEC) directly into test tubes containing 250 µL RLT plus lysis buffer, using a FACSAria (BD Biosciences). RNA was isolated using the RNeasy Plus Micro kit (Qiagen), according to the manufacturer’s instructions, including DNase digestion. qPCR was performed on an HT7900 system and analyzed as described above.
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