THP-1 cells were treated with 30 ng/mL phorbol-12-myristate 13-acetate (PMA, Sigma-Aldrich, Saint Luis, MO, USA) for 3 days, and induced into macrophage. EAST-6 antigen (1 μg/mL, BEI, Manassas, VA, USA) was incubated with antibody (EAST-6, Abcam, Massachusetts, USA) for 15 min, followed by incubation with dye (CF488A, Sigma-Aldrich, Saint Luis, MO, USA) for 15 min. THP-1 macrophage was then transfected (HiPerFect Transfection Reagent, Qiagen, MD, USA) with either nonsense sequence control (50 nM, Thermo Scientific, Waltham, MA, USA) or miR-23a-3p mimic (50 nM, Thermo Scientific) for 24 h, followed by treatment with ESAT6 antigen-antibody-CF488A compound. Phagocytosis was assayed by flowcytometry, using the CytomicsTM FC500 (Beckman Coulter). Specific ESAT6 antigen-antibody-CF488A up-take of each sample was expressed as a percentage of CF-488A (+) cells.
Cf488a
Manufactured by Merck Group
Sourced in United States
The CF488A is a fluorescent dye molecule developed by Merck Group for use in biological and biochemical research applications. It is designed to emit fluorescence in the blue-green region of the visible light spectrum when excited by a suitable light source. The CF488A dye can be conjugated to various biomolecules, such as antibodies, proteins, or nucleic acids, to enable fluorescent labeling and detection in various experimental techniques.
Automatically generated - may contain errors
Lab products found in correlation
Cytomics fc500, by Beckman Coulter (1 mentions)
Hiperfect transfection reagent, by Qiagen (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Tcs sp5, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Cf 568, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade with dapi, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Anti tau, by Cell Signaling Technology (1 mentions)
Anti vgat, by Synaptic Systems (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Anti vglut1, by Synaptic Systems (1 mentions)
Cf633, by Merck Group (1 mentions)
Nab228, by Merck Group (1 mentions)
Tau12, by BioLegend (1 mentions)
6 protocols using cf488a
1
Phagocytic Uptake of ESAT6 in THP-1 Macrophages
2
Quantitative Analysis of Immunofluorescence in Atherosclerosis
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Immunofluorescent assay was performed as previously described.13 , 14 , 17 , 18 , 19 Briefly, frozen tissue sections were labeled with appropriate isotype IgG control or antibodies as indicated in the figures and visualized with appropriate secondary antibodies conjugated with CF 488A or CF 568 (Sigma) for the unconjugated primary antibodies. Sections were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI; Sigma). Images were examined and acquired with SP5 confocal microscopy with Leica TCS Sp5 software (Leica) at room temperature. Positively stained areas for the respective antibody in a given image were highlighted and quantified (percentage over the atherosclerotic lesion area) by computer‐assisted quantification using the Axiovision software. Two experienced investigators blinded to the treatments each tissue had received performed this analysis. Three sections were analyzed per aortic roots (or per mouse) and averaged.
3
Quantifying Receptor Expression in Cells
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A total of 5 × 104 cells were plated on culture chambers (Sarstedt, Germany), fixed with 4% paraformaldehyde (Invitrogen, Waltham, MA, USA), and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA). Then, cells were blocked with goat serum and subsequently incubated with rabbit primary AR monoclonal antibody (MA5-13426; Sigma-Aldrich, St. Louis, MO, USA) and rabbit primary ERβ polyclonal antibody (PA1-310B; Invitrogen, Massachusetts, USA) overnight at 4 °C and with shaking. Then, cells were washed with PBS and incubated with secondary antibody (CF488A; Sigma-Aldrich, St. Louis, MO, USA). Finally, slides were mounted with Prolong Gold Antifade with DAPI (Invitrogen, Waltham, MA, USA). Images were captured with the Optika fluorescence microscope (Optika IM-3LD2 Microscope, Bérgamo, Italy). Immunofluorescence quantification was analyzed with the RGB value plugin available in ImageJ software 1.53e version.
4
Immunofluorescence Analysis of SPs-rPA Compositions
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Immunofluorescence analyses of SPs-rPA1+2, SPs-rPA3+4 and SPs-rPA83m compositions were performed as described previously [24 (link)]. Primary antibodies were used in 1/100 dilution; secondary anti-mouse IgG antibodies conjugated to Alexa Fluor® 546 (A-11030, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) or secondary anti-human IgG antibodies conjugated to CF™ 488A (SAB4600055, Sigma-Aldrich, St. Louis, MO, USA) were used in 1/200 or 1/100 dilutions, respectively.
5
Immunostaining and Synaptic Analysis of Autaptic Neurons
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Immunostaining was performed as previously reported50 (link),51 . After fixation, permeabilization and blocking, autaptic neurons cultures were incubated with the following primary antibodies: anti-MAP2 (guineapig polyclonal, Synaptic Systems, Goettingen, Germany, Cat. No. 188 004, 1:1000 dilution)50 (link), anti-VGLUT1 (rabbit polyclonal, Synaptic Systems, Cat. No. 135 302, 1:2000 dilution)50 (link), anti-VGAT (rabbit polyclonal, Synaptic Systems, Cat. No. 131 002, 1:1000 dilution), and anti-tau (mouse monoclonal, Cell Signaling Technology, Beverly, MA, USA, Cat. No. 4019, 1:1000 dilution). Then, samples were incubated with the appropriate species-specific fluorochrome-labelled goat secondary antibodies [Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA, Cat. No. A11073) for MAP2, Alexa Fluor 594 (Invitrogen, Cat. No. A11037) for VGLUT1 and VGAT, and CF 488A (Sigma-Aldrich, St. Louis, MO, USA, Cat. No. SAB4600042) for tau, 1:400 dilution]. Samples were mounted in mounting medium containing DAPI (ProLong Gold Antifade Mountant with DAPI, Invitrogen, Cat. No. P36931).
Confocal images of neurons were captured as previously reported51 . Synaptic puncta were analysed according to previously reported procedures50 (link)–52 (link). Dendritic and axonal morphologies were analysed using NeuronJ, which was accessed as an ImageJ plugin (v1.4.3, Erik Meijering).
Confocal images of neurons were captured as previously reported51 . Synaptic puncta were analysed according to previously reported procedures50 (link)–52 (link). Dendritic and axonal morphologies were analysed using NeuronJ, which was accessed as an ImageJ plugin (v1.4.3, Erik Meijering).
6
Oligomer Detection with Fluorescent Antibodies
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To detect oligomers in samples, we labeled the antibodies Nab228 (Sigma‐Aldrich) and Tau12 (BioLegend, San Diego, CA, USA) with the fluorescent dyes CF633 (Sigma‐Aldrich) and CF488A (Sigma‐Aldrich) according to manufacturer's protocol. The principle of reaction, the purification, and the determination of concentration and degree of labeling were previously described.
19 (link)
19 (link)
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