Neutrophils were cultured at 37°C, 5%CO2 in RPMI-1640 with L-glutamine and stimulated with Fas L (500 ng/ml), cycloheximide (1 nM, Sigma), staurosporine (1 μM), opsonized F. alocis (multiplicity of infection [MOI] 10, 50, 100), or opsonized heat-killed F. alocis (MOI 10). After 24 h of culture, cells were processed as cytospins and stained with a Hema-3 Kit or stained with APC Annexin V Apoptosis Detection Kit and 7-AAD (BioLegend, San Diego, CA, USA). Samples were read on a BD FACSCelesta flow cytometer and data were analyzed using FlowJo software (Ashland, OR, USA). In some experiments, neutrophils were pretreated with TLR6 neutralizing antibody (50 μg/ml, Invivogen) or isotype control (Rat IgG,50 μg/ml, Invivogen) for 60 min and/or TLR2 neutralizing antibody (50 μg/ml; clone TL2.1; BioLegend) or isotype control IgG2a kappa (50 μg /ml; clone MOPC-173; BioLegend) for 30 min. All experiments were done in media with no serum except for assays involving conditioned media or transwells where media was supplemented with 5% heat-inactivated fetal bovine serum.
Apc annexin 5 apoptosis detection kit and 7 aad
Manufactured by BioLegend
Sourced in United States
The APC Annexin V Apoptosis Detection Kit and 7-AAD is a laboratory equipment product that enables the detection and measurement of apoptosis in cells. Annexin V is a calcium-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine (PS), which is exposed on the outer membrane of apoptotic cells. The APC (Allophycocyanin) conjugated Annexin V binds to exposed PS, allowing for the identification of apoptotic cells. 7-AAD (7-Aminoactinomycin D) is a fluorescent dye that is used to stain and detect late apoptotic and necrotic cells. This kit provides the necessary reagents to perform these analyses.
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2 protocols using apc annexin 5 apoptosis detection kit and 7 aad
1
Neutrophil Apoptosis Induced by F. alocis
2
Neutrophil Apoptosis Induction and Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Neutrophils were cultured at 37°C, 5%CO2 in RPMI‐1640 with L‐glutamine and stimulated with Fas L (500 ng/ml), cycloheximide (1 nM, Sigma), staurosporine (1 μM), opsonized F. alocis (multiplicity of infection [MOI] 10, 50, 100), or opsonized heat‐killed F. alocis (MOI 10). After 24 h of culture, cells were processed as cytospins and stained with a Hema‐3 Kit or stained with APC Annexin V Apoptosis Detection Kit and 7‐AAD (BioLegend, San Diego, CA, USA). Samples were read on a BD FACSCelesta flow cytometer and data were analyzed using FlowJo software (Ashland, OR, USA). In some experiments, neutrophils were pretreated with TLR6 neutralizing antibody (50 μg/ml, Invivogen) or isotype control (Rat IgG,50 μg/ml, Invivogen) for 60 min and/or TLR2 neutralizing antibody (50 μg/ml; clone TL2.1; BioLegend) or isotype control IgG2a kappa (50 μg /ml; clone MOPC‐173; BioLegend) for 30 min. All experiments were done in media with no serum except for assays involving conditioned media or transwells where media was supplemented with 5% heat‐inactivated fetal bovine serum.
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