Tumors samples were frozen in optimum cutting temperature (Sakura Finetek, Torrance, CA, USA). Acetone-fixed cryosections (8 µm) were consecutively treated with 0.01% Triton X-100 for 15 min, 0.03% hyaluronidase for 15 min, FcR Block (Innovex Biosciences, Richmond, CA, USA) for 40 min, and Background Buster (Innovex Biosciences) for 20 min. The sections were then stained with primary antibodies diluted in PBS + 5% bovine serum albumin and 0.1% saponin for 1 h at room temperature, washed, and stained with the secondary antibodies at room temperature for 30 min. Nuclei were counterstained with DAPI (1 µg/ml) for 2 min. Primary antibodies included anti-hCD45 at 2 µg/ml (clone H130; BioLegend), FITC-conjugated anti-pan cytokeratin at 1:60 dilution (clone CK3-6H5; Miltenyi Biotec, Bergisch Gladbach, Germany), and AF647-conjugated anti-hCD8a at 1.25 µg/ml (clone RPA-T8; BioLegend). The secondary antibody included goat anti-mouse IgG1 AF568 at 1:2000 dilution (Thermo Fisher Scientific). For acquisition, data were acquired by sequential acquisition, and tile-scan imaging was performed on an SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany).
Clone ck3 6h5
Manufactured by Miltenyi Biotec
Sourced in Germany
The Clone CK3-6H5 is a cell line that produces a monoclonal antibody specific for the CD3 epsilon chain of the T cell receptor complex. This antibody can be used for the identification and isolation of T cells.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer z1, by Zeiss (6 mentions)
Goat serum, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (2 mentions)
Anti hcd45, by BioLegend (1 mentions)
Clone rpa t8, by BioLegend (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Cd45 pe clone hi30, by BD (1 mentions)
Triton x 100, by Research Products International (1 mentions)
Clone cam5, by BD (1 mentions)
Cd45 pe clone 30 f11, by BD (1 mentions)
Triton x 100, by Merck Group (1 mentions)
3 protocols using clone ck3 6h5
1
Immunofluorescent Staining of Frozen Tumor Sections
2
Enumeration of Circulating Tumor Cells
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Cells collected from 20 samples (18 patients) were fixed with 2% paraformaldehyde (Electron Microscopy Sciences) for 10 min, permeabilized with 0.4% v/v Triton X-100 (Research Products International Corp) for 7 min, blocked with 5% goat serum (Invitrogen) for 30 min, and labeled for 1 h at RT with DAPI (Life Technologies), anti CD45-phycoerythrin (CD45-PE, Clone HI30, BD Biosciences), and a cocktail of primary antibodies to identify CK positive cells (Pan-CK clone AE1/AE3, eBioscience, clone CK3-6H5, Miltenyi Biotec, and CK clone CAM5.2, BD Biosciences). PSA positive cells were identified using a rabbit anti-PSA polyclonal antibody (Dako) and were visualized using AlexaFluor-647 secondary antibodies (Anti-rabbit IgG, Cell Signaling). For three patient samples, PSA staining was replaced by Vimentin (Vimentin-AF467, clone V9, Abcam) and N-cadherin (NC-AF647, clone EPR1791-4, Abcam) staining. After staining, the cells were imaged (Axio Observer Z1, Zeiss) and manually enumerated using specific classification criteria (Fig. 3a, d ). For patient samples, purity is calculated as the number of CTCs that meet the criteria described in Fig. 3a (i.e., DAPI+ with CK and/or PSA positivity or nuclear size >9 μm with nuclear/cytoplasmic (N:C) ratio >0.6 and negative for CD45) divided by the total number of DAPI positive cells counted.
3
Multiparametric Profiling of Cell Populations
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After Vortex processing, cells were collected in a cell culture-treated 96-well plate or a PolySorp-coated eight-well strip (Nunc), fixed with 4% paraformaldehyde (Electron Microscopy Sciences) for 10 min, permeabilized with 0.4% v/v Triton X-100 (Sigma Aldrich) for 7 min, blocked with 10% goat serum (Invitrogen) for 30 min, and labeled for 1 h at room temperature (RT) with 4,6-diamidino-2-phenylindole (DAPI; Life Technologies), anti-CD45-phycoerythrin antibody (CD45-PE, clone HI30, BD Biosciences), and a cocktail of primary antibodies labeled with fluorescein isothiocyanate to identify cytokeratin (CK)-positive cells (clone CK3-6H5, Miltenyi Biotec, and clone CAM5.2, BD Biosciences). 28 For experiments with healthy mouse blood, a similar staining protocol was optimized but with a rat anti-mouse CD45-PE antibody (CD45-PE, clone 30-F11, BD Pharmingen). For patient-derived orthotopic xenograft (PDOX) samples, an anti-CK-AlexaFluor (AF) 488 antibody (clone AE1/AE3, eBioscience) and an antivimentin-AF647 antibody (clone V9, Abcam) were used in addition to the previously listed antibodies. After staining, the cells were imaged (Axio Observer Z1, Zeiss) and manually enumerated using specific classification criteria previously published. 27 Capture performance was evaluated as follows:
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