Monoclonal anti-mouse IgD (clone 1.3.7) was first dialyzed in 1x PBS over night at 4°C. 10 mg of the mAb (in 1ml of 0.1M NaHCO3, pH8.0) was incubated with 1 mg of the biotin reagent (EZ-Link Sulfo-NHS-LC-Biotin, Fisher Scientific, Pittsburg, PA) for 2 hours at room temperature. Labeled proteins were then dialyzed against normal saline. For the FITC labeling, 0.5 ml of mAb was prepared in 1×PBS containing 40 μl of the borate buffer (0.67M) and added to the FITC reagent (Pierce FITC Antibody Labeling Kit, Thermo Scientific, Rockford, IL). The reaction mixture was then incubated at room temperature for 60 minutes protected from light. Labeled proteins were purified with the purification resin provided with the kits.
Pierce fitc antibody labeling kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Pierce FITC Antibody Labeling Kit is a laboratory product that enables the conjugation of FITC (Fluorescein Isothiocyanate) to antibodies or other proteins. FITC is a fluorescent dye that can be used to label and detect the labeled proteins.
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Lab products found in correlation
Cyflow cube 6, by Sysmex (1 mentions)
Rtgf β1 ri, by Thermo Fisher Scientific (1 mentions)
Amicon ultra, by Merck Group (1 mentions)
Hiload 16 600 superdex 200 prep grade column, by GE Healthcare (1 mentions)
Ax10 imager a2, by Zeiss (1 mentions)
Hoechst 33342, by Beyotime (1 mentions)
Antifade mounting medium, by Beyotime (1 mentions)
Anti cd3 af700, by BioLegend (1 mentions)
10 protocols using pierce fitc antibody labeling kit
1
Biotinylation and FITC Labeling of Anti-mouse IgD
2
Quantification of CD24 and CD11b Expression
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A total of 1 × 106 leukocytes were used for each test and stained with 0.05 µg anti-CD24-FITC mAb (NS17), anti-CD11b-PerCp-Cy5.5 (rat IgG2b, clone M1/70, Abcam, Israel) or remained unstained for 30 min at RT. NS17 is a humanized IgG1 anti-CD24 antibody that we developed by means of genetic engineering and is fully characterized. The purified NS17 was labeled with FITC using the Pierce™ FITC Antibody Labeling Kit according to manufacturer’s instructions (ThermoFisher Scientific, Waltham, MA, USA).
After staining, the cells were washed twice with FACS buffer (0.01% sodium azide, 10% fetal bovine serum [FBS] in ice-cold PBS) and then analyzed by flow cytometry (CyFlow Cube 6, Sysmex, Germany). Data were analyzed following the creation of a hierarchical population tree in the software at the beginning of the screen. This template was used in all subsequent analyses. The template file included compensation adjustment, which was uniformly applied to all the collected data to minimize fluorescence overlap between detection channels. The percentage of positive cells was determined by subtracting the percentage of CD24 and CD11b-positive cells (dual stain) from CD24-positive cells (single stain). Flow cytometry representative graph of the different populations and their fluorescence intensity is detailed inFigure 1 .
After staining, the cells were washed twice with FACS buffer (0.01% sodium azide, 10% fetal bovine serum [FBS] in ice-cold PBS) and then analyzed by flow cytometry (CyFlow Cube 6, Sysmex, Germany). Data were analyzed following the creation of a hierarchical population tree in the software at the beginning of the screen. This template was used in all subsequent analyses. The template file included compensation adjustment, which was uniformly applied to all the collected data to minimize fluorescence overlap between detection channels. The percentage of positive cells was determined by subtracting the percentage of CD24 and CD11b-positive cells (dual stain) from CD24-positive cells (single stain). Flow cytometry representative graph of the different populations and their fluorescence intensity is detailed in
3
Recombinant HMGB1 Production and Modifications
4
Quantitative Analysis of Fluorescein Labeling
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The number of FL-5Mal moieties conjugated to NFG1CG4-hFasLECD was estimated according to the calculation equations described in the instruction manual of Pierce FITC Antibody Labeling kit (Thermo Fisher Scientific Inc. 2014 ) from the measured absorbance values at 280 and 495 nm. Molar extinction coefficient of Fluorescein group at 495 nm and that of NFG1CG4-hFasLECD at 280 nm were assumed as 70,000 and 29,005, respectively. The latter value was obtained using the Prot Param tool on the ExPASy Server (Gasteiger et al. 2005 ).
5
HMGB1 Purification and Labeling
6
EGFR-Targeting Phage Binding Assay
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Phages expressing peptide sequences (01cys_EGFR, 04cys_EGFR, 06cys_EGFR, and 30cys_EGFR) and a wild-type M13 phage were conjugated to fluorescein isothiocyanate (FITC) in order to analyze their binding affinity to EGFR on MDA-MB-231 by flow cytometry. The “Pierce FITC Antibody Labeling Kit” (cat #53027—Thermo Fisher Scientific) was used for the conjugation following the manufacturer’s instructions.
7
Conjugation of rTGF-β1 RI with DMPE-PEG-maleimide
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DMPE-PEG-maleimide was purchased from Nanocs, Inc. (NY, USA); recombinant rTGF-β1 RI and TGF-β1 were purchased from R&D Inc. (MN, USA). The recombinant rTGF-β1 RI contains ten cysteines that can react with maleimide. The rTGF-β1 RI cysteine residues are active sites for conjugation and are not involved in binding to TGF-β1. DMPE-PEG-maleimide was dissolved in PBS (pH 7.4) to a final concentration of 1 mg/mL, and rTGF-β1 RI was prepared according to the manufacturer’s instructions. DMPE-PEG-maleimide was mixed with rTGF-β1 RI (molar ratio of 10–20:1) and incubated for 2 h at room temperature. The mixed solution was then filtered using Amicon Ultra (Ultracel-10 kDa, Millipore, IL, USA) to remove impurities and stored at − 80 °C for later use. For confocal imaging and FACS analysis of rTGF-β1 RI binding efficiency, rTGF-β1 RI proteins were prelabeled with FITC according to the manufacturer’s instructions (Pierce™ FITC Antibody Labeling Kit, Thermo Fisher Scientific), and then FITC-labeled rTGF-β1 RI was incubated with DMPE-PEG-maleimide to afford a DMPE-PEG-rTGF-β1 RI-FITC solution. TGF-β1 protein was labeled with Cy3 according to the manufacturer’s instructions (Lightning-Link™ Cy3 Kit, Innova Biosciences).
8
Purification and FITC Labeling of EBOV GP
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Recombinant EBOV GP was produced from insect cells and purified using immunoaffinity chromatography following previously published methods (Lehrer et al., 2017 (link)). To further increase the level of purity, protein used for our studies was subjected to an additional purification step via size-exclusion chromatography using a HiLoad 16/600 Superdex 200 prep grade column (GE Healthcare Life Sciences, Piscataway, NJ) equilibrated in phosphate buffered saline, pH7.4 (Figure 1A ). FITC labeling of purified EBOV GP was performed using the Pierce FITC Antibody Labeling Kit (Thermo Fisher Scientific) according to manufacturer's instructions. Briefly, protein prepared in borate buffer (50 mM sodium borate, pH 8.5) was added to FITC reagent and mixed by pipetting up and down. After incubation for 60 min at room temperature, the labeling reaction was added to the spin column containing a purification resin to remove unbound FITC. After thorough mixing, the purified FITC-labeled protein was eluted by centrifugation of the spin columns for 30–45 s at 1,000 g.
9
TRAIL Protein Labeling and Visualization
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The Pierce FITC Antibody Labeling kit (Thermo Fisher Scientific, Inc.) was used to label TRAIL-Mu3 or TRAIL according to the manufacturers instructions. PANC-1 cells (5×104/ml) were seeded in 24-well plates that contained slides and then incubated with labeled TRAIL-Mu3 or TRAIL. After 1 h at 37°C, cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. Cells were then stained with DiI (Beyotime Institute of Biotechnology) for 10 min and Hoechst 33342 (Beyotime Institute of Biotechnology) for 5 min at room temperature. Antifade mounting medium (Beyotime Institute of Biotechnology) was used to mount the cells, and the samples were analyzed by fluorescence microscopy (magnification, ×400; AX10 Imager A2; Carl Zeiss AG).
10
Fluorescent Labeling of Human IgA for Leukocyte Binding
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Human IgA purified from post OIT sera was fluorescently labeled using the Pierce FITC antibody labeling kit (Thermofisher Scientific, Rockford, IL) according to the manufacturer’s instructions. For IgA binding experiments, donor whole blood was stained with labeled IgA, along with antibodies to human leukocyte markers PE anti-CCR3, APC anti-FcεR1, AF700 anti-CD3, PE-Cy7 anti-CD16, BV510 anti-CD14 all from (Biolegend, San Diego, CA) for 30 minutes at 37°C.
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