RBCs, μEGs and nEGs were assayed for PS exposure using annexin V labeled with Alexa Fluor488 (AF488) (Invitrogen, Carlsbad, CA). Prior to analysis, each sample was incubated for 30 minutes at room temperature with annexin V conjugate in the presence of a binding buffer, which contained 10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2 (pH 7.4). Fluorescence was measured using a BD LSRII flow cytometer (excitation laser: 488 nm, emission filter: 515–545 nm), and the PS-positive populations were quantified using FlowJo V10.
Alexa fluor 488 af488
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488 (AF488) is a fluorescent dye used in various biomedical applications. It is a bright, photostable, and pH-insensitive fluorescent label that can be conjugated to different biomolecules, such as antibodies, proteins, and nucleic acids. AF488 has an excitation maximum at 495 nm and an emission maximum at 519 nm, making it suitable for detection using common fluorescence instrumentation.
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Lab products found in correlation
Zeba spin desalting column, by Thermo Fisher Scientific (3 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
A5316, by Merck Group (1 mentions)
Sc 13116, by Santa Cruz Biotechnology (1 mentions)
Mouse monoclonal antibody against cox 4, by Abcam (1 mentions)
Horseradish peroxidase, by Jackson ImmunoResearch (1 mentions)
Tris acetate gel, by Thermo Fisher Scientific (1 mentions)
Trypsinogen, by Merck Group (1 mentions)
Enterokinase, by Merck Group (1 mentions)
Ex cell 293 medium, by Merck Group (1 mentions)
Nickel nta, by Qiagen (1 mentions)
P 10 desalting column, by GE Healthcare (1 mentions)
Polyacrylamide desalting column, by Thermo Fisher Scientific (1 mentions)
Atto647n, by ATTO-TEC (1 mentions)
Horseradish peroxidase hrp, by Thermo Fisher Scientific (1 mentions)
Ma5 15739, by Thermo Fisher Scientific (1 mentions)
Protein g sepharose, by Thermo Fisher Scientific (1 mentions)
Donkey anti goat secondary antibody, by Thermo Fisher Scientific (1 mentions)
Goat anti human igg fc, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg, by Merck Group (1 mentions)
20 protocols using alexa fluor 488 af488
1
Annexin V-Based Phosphatidylserine Assay
2
Immunofluorescence and Western Blot Protocols
3
Fabrication of Porous Polymer Microfluidics
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TPE (CFS Fibreglass, UK) was prepared by mixing it with its polymerization catalyst, methyl ethyl ketone peroxide (MEKP), and polyethylene terephthalate, PET, (Daedong Polymer, South Korea) was used as substrate. The mixture of ethylene diacrylate (EDA, monomer) 0.485 g, methyl methacrylate (MMA, cross-linker) 0.485 g and benzophenone (BP, photo-initiator) 0.03 g was prepared for the grafting layer. Butyl methacrylate (BuMA, monomer) 0.6 g, ethylene dimethacrylate (EDMA, cross-linker) 0.4 g, 1-dodecanol (progen) 1.5 g and 2,2’-dimethoxy-2-phenylacetophenone (DMPAP, photo-initiator) 0.01 g were mixed for the monolithic porous polymer.
For droplet experiments, a 10% (v/v) mixture of fluorocarbon oil, FC-70 (3 M Fluorinert, USA), and 1H, 1H, 2H, 2H-perfluorooctanol (PFO, Sigma-Aldrich) was used for the continuous phase. The mobile phase was a mixture of 26 mM phosphate buffer (pH 7) and methanol (HiPerSolve for HPLC, BDH Prolabo) in a ratio of 5: 95 (v/v)
Fluorescein isothiocyanate, FITC, (Sigma-Aldrich, USA) and Alexa Fluor® 488, AF 488, (Invitrogen, USA) were diluted to 0.02 μM and 50 μM concentration in the mobile phase as the analytes.
For droplet experiments, a 10% (v/v) mixture of fluorocarbon oil, FC-70 (3 M Fluorinert, USA), and 1H, 1H, 2H, 2H-perfluorooctanol (PFO, Sigma-Aldrich) was used for the continuous phase. The mobile phase was a mixture of 26 mM phosphate buffer (pH 7) and methanol (HiPerSolve for HPLC, BDH Prolabo) in a ratio of 5: 95 (v/v)
Fluorescein isothiocyanate, FITC, (Sigma-Aldrich, USA) and Alexa Fluor® 488, AF 488, (Invitrogen, USA) were diluted to 0.02 μM and 50 μM concentration in the mobile phase as the analytes.
4
Collagen Cleavage Kinetics Analysis
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We studied cleavage kinetics of the mutant and normal control collagens by recombinant human MMP1 (gift of Prof. H. Nagase, Imperial College, London), the catalytic domain of MMP1 (Enzo Life Sciences), and MMP2 (EMD Biosciences). MMP1 was activated with 1 mM 4-aminophenylmercuric acetate [34 (link)]. For these experiments, purified type I collagen from the medium of either control cells or cells from the patient was fluorescently labeled either by AlexaFluor 488 (AF488, Invitrogen) or Cy5 (GE Healthcare) [29 (link)]. Binary mixtures of mutant-AF488/control-Cy5 and mutant-Cy5/control-AF488 in 50 mM Tris, 150 mM NaCl, 10 mM CaCl2, 0.05% Brij were prepared and co-processed with the enzyme at different temperatures. The cleavage products and kinetics were examined on precast 3–8% Tris-Acetate gels (Invitrogen) using the FLA5000 scanner (Fuji Medical Systems) for fluorescence detection and ScienceLab software supplied with the scanner for quantitative analysis.
5
Granzyme Purification and Protease Assays
6
Site-specific Labeling of Proteins
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Site-specific labeling of αS was performed in an αS variant with a single engineered cysteine at position 122 (αS N122C). This variant was expressed and purified as described above but including 5 mM DTT in all purification steps. The protein was labeled with maleimide-modified Alexa Fluor 488 (AF488) (Invitrogen, Carlsbad, USA) for 15–20 h at 4 °C in the dark. After quenching the reaction with 10 mM DTT, free unreacted dye in the protein solution was subsequently separated using a P10 desalting column (GE Healthcare, Waukesha, USA), and the labeled protein solution was flash frozen with liquid nitrogen and stored at −80 °C. The different peptides, PSMα3, dPSMα3, and LL-37, were labeled at a single engineered cysteine at the N-terminus with maleimide-modified Atto647N (ATTO-TEC, Siegen, Germany). The same labeling and purification strategy were followed as for αS, although in this case the unreacted free dye was removed from the protein solution using a polyacrylamide desalting column (Thermo Fisher Scientific, Waltham, USA). Two cleaning steps were required to remove completely the free dye from the labeled peptide solution.
7
Immunological detection of NSDV proteins
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A polyclonal rabbit-derived in-house antiserum was used to stain NSDV nucleoprotein (NP) in western blot (WB; 1:1000) and immunofluorescence analyses (1:200). This hyperimmune serum was collected from a rabbit after three consecutive immunizations with CCHFV NP (Kosova Hoti isolate) and confirmed to cross-react with NSDV NP in the respective assays. Monoclonal anti-ß actin antibodies (MA5–15739, Invitrogen) and rabbit-derived anti-membrane bound transcription factor protease site 1 (MBTPS1) antibodies (PA5–77103, Invitrogen) were diluted in 1:5000 and 1:1000 for WB analysis, respectively. Immunoprecipitation of HA-tagged proteins was performed using HA-specific H6908 antibodies (Sigma-Aldrich; 1:500). Precipitated proteins were detected in WB with monoclonal HA.11 antibodies (#901501, Biolegend; 1:1000). Secondary anti-mouse and anti-rabbit antibodies conjugated either with Horseradish peroxidase (HRP) or Alexa Fluor 488 (AF488; both Invitrogen) were used in WB (1:5000) or IFA (1:500), respectively.
8
Monoclonal Antibody Characterization and Labeling
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Monoclonal antibodies b96.11 and b78 were derived from a patient with autoimmune polyendocrine syndrome type 1 and recognize epitopes located at amino acid residues 308-365 and 451-585 respectively. 28 , 29 Mouse monoclonal antibody AE6D9 recognizes epitopes located at the A chain of insulin. 30 Human monoclonal antibody HAA1 (ATCC Manassas VA, USA; ATCC number: HB-8534) is directed against Blood group A antigen and served as an isotype control for mAb b96.11 and b78. B-cell lines and hybridoma were grown under standard conditions, and the antibodies were purified from cell supernatant using Protein G Sepharose (Catalog number 101242, Invitrogen). Monoclonal antibodies b78 and HAA1 were labelled with Alexa Fluor 647 (AF 647) (Catalog number A20173, Invitrogen), while b96.11 and AE6D9 were labelled with Alexa Fluor 488 (AF 488) (Catalog number A20181, Invitrogen) according to the manufacturer's instructions. TGN-38 antibody (2F7.1) (catalog number NB300-575SS) was obtained from Novus Biologicals (Littleton, CO).
9
Quantifying RSV Infection in Lung Cell Lines
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HEp-2, A549 and BEAS-2B cells were seeded at a concentration of 17,500 cells/well in black CELLSTAR®® 96 well plates with a µclear®® flat bottom suitable for fluorescence microscopy (Greiner-bio one) one day prior to inoculation. Briefly before inoculation, the cells were washed with DMEM-0. Clinical RSV and RSV A2 were diluted to infect the cells at a multiplicity of infection (MOI) of 0.01. Virus was left to adhere for 2 h at 37 °C, 5% CO2 and replaced with DMEM-10. Cells were fixed with 4% paraformaldehyde after 24 h, 48 h and 72 h, permeabilized with triton X-100 and stained with polyclonal goat-anti-RSV antibody (Virostat; specificity all viral antigens) followed by donkey-anti-goat secondary antibody conjugated with Alexa Fluor 488 (AF488) (Thermo Fisher Scientific) and additional DAPI nucleus staining (Sigma-Aldrich).
10
Primary Mouse Monoclonal IgG2a Anti-gpK8.1
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