Surface Labeling 2G12 was previously described [47 (link)]. Infected primary CD4 T cells were treated with compound 24 h post infection. 48 h post infection, cells were incubated for 20 min at 37 °C, with 5 μg/mL 2G12 (AB002; Polymun). Cells were then washed once with PBS and incubated with 1 μg/mL anti-human (Alexa Fluor 647; Invitrogen, Waltham, MA, USA) secondary Abs and the viability dye AquaVivid (Thermo Fisher Scientific, Waltham, MA, USA) for 15 min in PBS. Cells were washed again with PBS and fixed in a 2% PBS-formaldehyde solution. Infected cells were stained intracellularly for HIV-1 p24, using a Cytofix/Cytoperm fixation/permeabilization kit (BD Biosciences, Mississauga, ON, Canada) and fluorescent anti-p24 MAb (phycoerythrin [PE]-conjugated anti-p24, clone KC57; Beckman Coulter/Immunotech). The percentage of infected cells (p24+) was determined by gating the living cell population on the basis of viability dye staining (AquaVivid; Thermo Fisher Scientific). Samples were acquired on an LSR II cytometer (BD Biosciences), and data analysis was performed using FlowJo vX.0.7 (Tree Star, Ashland, OR, USA).
Anti human alexa fluor 647
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Anti-human (Alexa Fluor 647) is a fluorescent-labeled antibody used for the detection and analysis of human proteins or cells. It is conjugated with the Alexa Fluor 647 dye, which emits light in the far-red/near-infrared spectrum when excited. This product can be used in various immunoassay techniques, such as flow cytometry, immunohistochemistry, and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Lsrii cytometer, by BD (5 mentions)
Cytofix cytoperm fixation permeabilization kit, by BD (4 mentions)
Aquavivid, by Thermo Fisher Scientific (2 mentions)
Aquavivid viability dye, by Thermo Fisher Scientific (1 mentions)
293fectin, by Thermo Fisher Scientific (1 mentions)
Live dead aqua fixable cell stain, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Cellfix, by BD (1 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (1 mentions)
Fcr block, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
7 protocols using anti human alexa fluor 647
1
Quantifying HIV-1 Infection Using 2G12 Labeling
2
HIV-1 Cell Surface Binding Assay
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Cell-surface staining was performed as previously described [15 (link),16 (link),17 (link)]. Primary CD4+ T cells were isolated from healthy donors and infected with HIV-1CH58TF, HIV-1JRFL, or HIV-1AD8. Binding of HIV-1-infected cells via plasma (1:1000 dilution) or 17b mAb (5 μg/mL) in the presence or absence of 50 μM compounds was performed 48 h after infection. Cells were then incubated at 37 °C for 1 h followed by adding anti-human Alexa Fluor 647 (Invitrogen, Waltham, MA, USA) secondary Abs for 20 min. Cells were then stained intracellularly for HIV-1 p24, using the Cytofix/Cytoperm Fixation/ Permeabilization Kit (BD Biosciences, Mississauga, ON, Canada) and the fluorescent anti-p24 mAb (PE-conjugated anti-p24, clone KC57; Beckman Coulter/Immunotech). The percentage of infected cells (p24+ cells) was determined by gating the live cell population on the basis of the AquaVivid viability dye (Thermo Fisher Scientific, Waltham, MA, USA) staining. Samples were analyzed on an LSRII cytometer (BD Biosciences, Bad Wildbad, Germany), and data analysis was performed using FlowJo vX.0.7 (Tree Star, Ashland, OR, USA).
3
HIV-1 Infected Cell Binding Assay
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Cell-surface staining was performed as previously described [15 –17 (link)]. Primary CD4 T cells were isolated from healthy donors and infected with HIV-1CH58TF, HIV-1JRFL or HIV-1AD8. Binding of HIV-1-infected cells by plasma (1:1,000 dilution) or 17b mAb (5μg/ml) in the presence or absence of 50 μM compounds was performed 48 hours after infection. Cells were then incubated at 37 °C for 1 hour followed by adding anti-human Alexa Fluor-647 (Invitrogen) secondary Abs for 20 minutes. Cells were then stained intracellularly for HIV-1 p24, using the Cytofix/Cytoperm Fixation/ Permeabilization Kit (BD Biosciences, Mississauga, ON, Canada) and the fluorescent anti-p24 mAb (PE-conjugated anti-p24, clone KC57; Beckman Coulter/Immunotech). The percentage of infected cells (p24+ cells) was determined by gating the live cell population on the basis of the AquaVivid viability dye staining. Samples were analyzed on an LSRII cytometer (BD Biosciences), and data analysis was performed using FlowJo vX.0.7 (Tree Star, Ashland, OR, USA).
4
Flow Cytometric Analysis of Env-mediated Cell-surface Staining
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Cell-surface staining was performed as previously described [52 (link)]. Infected CEM-NKR cells were incubated with anti-Env antibodies (5 μg/ml) 48 h after infection. Cells were then incubated at 37 °C for 1 h followed by adding anti-human Alexa Fluor-647 (Invitrogen) secondary Abs for 20 min. The percentage of infected cells (GFP+) was determined by gating the live cell population on the basis of the AquaVivid viability dye staining. Samples were analyzed on an LSRII cytometer (BD Biosciences), and data analysis was performed using FlowJo vX.0.7 (Tree Star, Ashland, OR, USA). The results represent the average of the samples tested in triplicate.
5
Quantifying HIV-1 Infection via Surface Labeling
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Surface Labeling 2G12 was previously described (42) . Infected primary CD4 T cells were treated with compound 24 hours post infection. 48 hours post infection, cells were incubated for 20 min at 37℃, with 5 µg/mL 2G12 (AB002; Polymun). Cells were then washed once with PBS and incubated with 1 ug/ml anti-human (Alexa Fluor 647; Invitrogen) secondary Abs and the viability dye AquaVivid (Thermo Fisher Scientific) for 15 min in PBS. Cells were washed again with PBS and fixed in a 2% PBS-formaldehyde solution. Infected cells were stained intracellularly for HIV-1 p24, using a Cytofix/Cytoperm fixation/permeabilization kit (BD Biosciences, Mississauga, ON, Canada) and fluorescent anti-p24 MAb (phycoerythrin [PE]conjugated anti-p24, clone KC57; Beckman Coulter/Immunotech). The percentage of infected cells (p24 + ) was determined by gating the living cell population on the basis of viability dye staining (AquaVivid; Thermo Fisher Scientific). Samples were acquired on an LSR II cytometer (BD Biosciences), and data analysis was performed using FlowJo vX.0.7 (Tree Star, Ashland, OR, USA).
6
Epitope Mapping of P2X4 Antibodies
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Example 7
To determine the epitope to which the P2X4 functional antibodies bind the following mutations were generated in human P2X4; E95Q, V105M, G114D, A122V, S131N, A151P, G154R, L303P, N306K. DNA vectors containing huP2X4 sequences with these changes were generated using standard molecular biology techniques. DNA vectors were transfected into HEK293F cells using 293-fectin (Life Technologies 12347019) following the manufacturers guidelines. Cells expressing the huP2X4 variants were incubated with Antibody Nos. 1, 11, 29, and 33 together with the anti-human AlexaFluor 647 (Life Technologies A21445) detection reagent. Binding was measured using the FMAT plate reader. Variant S131N was shown to be important for the binding of Antibody Nos. 11, 29, and 33.
7
Flow Cytometric Analysis of SARS-CoV-2 Infection
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Cells were dislodged by washing in PBS and treatment with 0.05% trypsin (Gibco) for 20 min, followed by pipetting and centrifugation. For all subsequent steps, reagents were diluted in FACS buffer (PBS, 1% FCS, 2 mM EDTA) unless stated otherwise, and washed twice with FACS buffer between steps. Cells were stained with Live/Dead Fixable Aqua Cell Stain (1:200 in PBS, Life Technologies, L34957) combined with FcR block (1:200 in PBS, eBioscience), fixed in 4% formaldehyde (10 min at room temperature), and permeabilised in PBS containing 0.1% Triton-X (20 min at room temperature). Cells were incubated with antibodies against human MxA (clone M143, kind gift from G Kochs) and SARS-CoV-2 N protein (clone EY-2A, kind gift from Alain Townsend59 (link); 1:200, 30 min, 4 °C), and goat anti-mouse AlexaFluor 488 (Life Technologies, A11029) and anti-human AlexaFluor 647 (1:500, 30 min, 4 °C; Life Technologies, A21445), and resuspended in CellFix (1:10 in water; BD, 340181). Cells were analysed by flow cytometry on an Attune NxT Flow Cytometer (Thermo Fisher Scientific) and data were analysed using FlowJo software (BD).
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