Alexa fluor 647 monoclonal antibody labeling kit

The protocol for cytokine production and intracellular accumulation for flow cytometric detection was adopted from C. L. Fellman et al. and modified.[46 (link)] Cells were treated with 25 ng/ml Phorbol-12-Myristate-13-Acetate (PMA, Sigma-Aldrich) and 500 ng/ml ionomycin (Sigma-Aldrich). After 3 hours of incubation, 1 μg/ml Brefeldin-A (Sigma-Aldrich) was added and cells were cultured for additional 3 hours. Cells were then washed, stained with a viability dye (Fixable Viability Dye eFlour^®780, eBioscience), fixed and permeabilized (Foxp3/Transcription factor fixation/permeabilization concentrate and diluent, eBioscience) and stained with the following primary conjugated antibodies: anti-canine CD3-AlexaFluor488 (clone CA17.2A12, Leukocyte antigen biology lab, UCD), anti-canine CD4-PE (CA13.1E4, Leukocyte antigen biology lab, UCD) and anti-human IL17-AlexaFluor647 (Goat anti-human IL17, R&D systems). Anti-IL17 antibody was conjugated using Alexa Fluor 647 monoclonal antibody labeling kit (Invitrogen, Carlsbad, CA) per manufacturer’s instructions. Fluorescence was detected by a flow cytometer (Cytomics FC500, Beckman Coulter) and flow cytometry data were analyzed using FlowJo flow cytometry software (Tree Star Inc.).

The effect of JNJ-53718678 on stability of the RSV F protein was tested in a heat-shock Flow Cytometry assay. The full-length wild-type RSV F protein was transiently expressed in HEK293T cells. Forty-eight hours post transfection, the cells were detached using an EDTA-containing buffer and the cell suspension with or without added compounds was heat-shocked for 10 min at 55 °C. The cells were stained with the CR9501 and CR9503 antibodies (1 µg ml⁻¹) directly labeled with AlexaFluor647 (Alexa Fluor® 647 Monoclonal Antibody Labeling Kit, Molecular Probes/Invitrogen). Propidium iodide (Molecular Probes/Invitrogen) was used as a live-dead stain. Flow Cytometry was performed on FACS Canto II instrument (BD Biosciences). The data were analyzed using FlowJo9.6.1 software. A single cell population was selected on the side and forward scatters dot-plot. Within this population, PI-negative cells were analyzed for staining with CR9501 and CR9503 antibodies. Mean fluorescence intensity was calculated and the values of the heat-shocked samples were normalized to untreated (37 °C) samples.

Cells were harvested by trypsinization, washed and resuspended in FACS buffer (PBS, 1% FBS, pH 7.4). Cell surface antigen density (copy number per cell) measurement was performed as described^{34 (link)}. Briefly, primary anti-guide or effector antibody was fluorophore-labeled using the Alexa Fluor^® 647 Monoclonal Antibody Labeling Kit (Molecular Probes). We measured the effective Fluorophore/Protein (F/P) ratio of each Alexa Fluor^® 647-conjugated antibody using Simply Cellular^® anti-Human IgG (Bang’s Laboratory) according to manufacturer’s instructions. Following incubation with labeled antibody and PBS washing, cells were analyzed by BD Accuri C6 flow cytometer (BD Biosciences). MFI (Median Fluorescence Intensity) values were converted into Molecules of Equivalent Soluble Fluorochrome (MESF) using Quantum™ Alexa Fluor^® 647 MESF (Bang’s Laboratory) according to manufacturer’s recommendations. MESF was converted into Antibody Binding Capacity (ABC) using the F/P ratio determined above^{34 (link)}.

The purified anti-MyHC1, anti-MyHC2A, anti-MyHC2X, and anti-MyHC2B antibodies were conjugated to the Alexa Fluor 647, Alexa Fluor 350, Fluorescein, and AnaTag™ HiLyte™ Fluor 594 fluorophores, respectively. Conjugation reactions were carried out according to the manufacturer’s instructions for the Alexa Fluor 647 Monoclonal Antibody Labeling Kit (Life Technologies, Carlsbad, CA, USA), the Alexa Fluor 350 Antibody Labeling Kit (Life Technologies), the Fluorescein Labeling Kit-NH₂ (Dojindo Laboratories, Kumamoto, Japan), and the AnaTag™ HiLyte™ Fluor 594 Microscale Protein Labeling Kit (AnaSpec, Inc., Fremont, CA, USA).

The MW1 antibody recognizing an epitope within the polyQ stretch of HTT was developed by Paul Patterson [27] (link) and obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. 2B7 antibody binds to the N17 region of HTT; its generation and characterization were described previously [32] (link). 4C9 antibody was raised against the human PRR region in exon1 of the HTT protein [66] (link). 3B5H10 antibody is specific for the polyQ stretch of HTT and was generated and characterized by Steve Finkbeiner [67] (link), [68] (link). 2B7, 4C9 and MW1 antibodies were obtained from the CHDI Foundation (New York, NY). Antibodies MAB2166 and 3B5H10 were obtained from a commercial source (Millipore cat. n. MAB2166 and Sigma cat. n. P1874, respectively). Custom terbium cryptate and D2-fluorophore antibody labeling was performed by CisBio (Bagnols, France). Depending on the batch used, antibodies were cross-linked to 5 to 7 mol of terbium cryptate or D2-fluorophore per mole of antibody. Alexa-647 labeled 2B7 antibody was generated using the Alexa Fluor 647 Monoclonal Antibody Labeling Kit from Life Technologies (cat.n. A20186) as per manufacturer's instructions. Antibody against GAPDH was from Sigma (cat.n. G9545).