We performed a cell-based coronavirus immunostaining assay using a rabbit (2019-nCoV) spike S1 antibody (1:50; Sino Biological, Duesseldorfer, Germany) and goat anti-rabbit secondary antibody conjugated with Alexa Fluor 647 (A-21245; 1:1000; Invitrogen, Carlsbad, CA, USA), as previously described (Stefanik et al., 2021 (link)).
Alexa fluor 647 a 21245
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 647 (A-21245) is a fluorescent dye produced by Thermo Fisher Scientific. It is a far-red fluorescent dye with excitation and emission maxima at 650 nm and 665 nm, respectively. The dye can be used for various applications in life science research, including fluorescence microscopy, flow cytometry, and immunoassays.
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Lab products found in correlation
Collagenase type 2, by Thermo Fisher Scientific (2 mentions)
Facsaria fusion flow cytometer, by BD (2 mentions)
2019 ncov spike s1 antibody, by Sino Biological (1 mentions)
Alexa fluor 700, by BioLegend (1 mentions)
Brilliant violet 421, by BioLegend (1 mentions)
Ter119, by BioLegend (1 mentions)
Rb6 8c5, by BioLegend (1 mentions)
Brilliant violet 605, by BD (1 mentions)
Ra3 6b2, by BioLegend (1 mentions)
4 protocols using alexa fluor 647 a 21245
1
Cell-Based Coronavirus Immunostaining Assay
2
Multicolor Flow Cytometry Panel
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The following antibodies were purchased from BioLegend and used at a 1:400 dilution unless indicated otherwise: anti-CD45.1/Ly5.1 (APC-Cy7, A20), anti-CD45.2/Ly5.2 (Alexa Fluor 700, 104), anti-CD3e (FITC, 145-2C11), anti-CD11b/Mac-1 (1:1,600, FITC or PerCP-Cy5.5, M1/70), anti-Ly6C/Ly6G (1:1,600, FITC or PerCP-Cy5.5, RB6-8C5), anti-CD45R/B220 (FITC or APC, RA3-6B2), anti-Ter119 (FITC, Ter-119), anti-CD117/c-kit (Brilliant Violet 421 (1:600) or PE; BioLegend, or APC-H7, 2B8, (1:200) BD Bioscience), anti-Sca-1 (Pe-Cy7, E13-161.7), anti-CD48 (1:800, PerCP-Cy5.5, HM48-1), anti-CD150 (1:600, PE-Dazzle or 1:600 Brilliant Violet 605, TC15-12F12.2), anti-CD135/Flk2 (1:200, PE, A2F10.1, BD Pharmingen), anti-CD34 (1:30, Alexa Fluor 700 RAM34, eBioscience), anti-Ki67 (1:200, Alexa Fluor 647, 11F6), anti-CD201 (1:200, EPCR, PE anti-mouse, RCR16), anti-p-IRF3 (1:25, S396, D601M, rabbit monoclonal antibody 29047, Cell Signaling), goat anti-rabbit secondary (1:500, Alexa Fluor 647, A21245, Invitrogen), anti-γH2AX (1:100, Alexa Fluor 647 (Ser 139), 2F3) and anti-p65 (1:100, Alexa Fluor 488, p65, Santa Cruz Biotechnologies) antibodies.
3
Isolation and Purification of Rat Choroid Plexus Cells
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Choroid plexus was isolated from 10 male rats, minced, and digested in collagenase (15 mg/ml collagenase (type II, Gibco®, Grand island, NY, USA) in artificial CSF (aCSF)-HEPES containing (in mM): 120 NaCl, 2.5 KCl, 3 CaCl2, 1.3 MgSO4, 1 NaH2PO4, 10 glucose, 17 Na-HEPES, pH 7.56 for 30 min at 37 °C in a table shaker at 800 rpm. The supernatant was removed after 5 min centrifugation (600×g) and the pelleted cells were resuspended in aCSF-HEPES. The cells were triturated 20 times with a 1000 µl pipette and filtered through a 70 μm filter (pluriStrainer, Mini 70 µm, PluriSelect, Leipzig, Germany), prior to incubation with an anti-NKCC1 antibody with an extracellular epitope (1:200 in aCSF-HEPES, #ANT-071, Alomone Labs™, Jerusalem, Israel) for 30 min at 4 °C. The cells were pelleted (600 × g, 5 min) and resuspended in secondary antibody (1:500 in aCSF-HEPES, Alexa Fluor® 647—A-21245, Invitrogen™, Carlsbad, California, USA), in which it was kept for 20 min at 4 °C prior to centrifugation (600×g, 5 min) and resuspension in cold aCSF-HEPES. Cells were analyzed and sorted on a FACSAria Fusion flow cytometer (BD Biosciences, Lyngby, Denmark).
4
Isolation and Characterization of Choroid Plexus Cells
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Choroid plexus was isolated from 10 male rats, minced, and digested in collagenase (15 mg/ml collagenase (type II, Gibco®, Grand island, NY, USA) in artificial CSF (aCSF)-HEPES containing (in mM): 120 NaCl, 2.5 KCl, 3 CaCl2, 1.3 MgSO4, 1 NaH2PO4, 10 glucose, 17 Na-HEPES, pH 7.56 for 30 min at 37°C in a table shaker at 800 rpm. The supernatant was removed after 5 min centrifugation (600×g) and the pelleted cells were resuspended in aCSF-HEPES. The cells were triturated 20 times with a 1000 µl pipette and filtered through a 70 μm filter (pluriStrainer, Mini 70 µm, PluriSelect, Leipzig, Germany), prior to incubation with an anti-NKCC1 antibody with an extracellular epitope (1:200 in aCSF-HEPES, #ANT-071, Alomone Labs™, Jerusalem, Israel) for 30 minutes at 4°C. The cells were pelleted (600×g, 5 min) and resuspended in secondary antibody (1:500 in aCSF-HEPES, Alexa Fluor® 647 -A-21245, Invitrogen™, Carlsbad, California, USA), in which it was kept for 20 min at 4C prior to centrifugation (600×g, 5 min) and resuspension in cold aCSF-HEPES. Cells were analyzed and sorted on a FACSAria Fusion flow cytometer (BD Biosciences, Lyngby, Denmark).
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