Human peripheral blood mononuclear monocytes were isolated as previously described (van Hemert et al., 2010 ) with modifications. Buffy coats from peripheral blood of three healthy donors were obtained from the Sanquin Blood Bank, Nijmegen, The Netherlands. Isolated PBMCs were washed and resuspended in Iscove's Modified Dulbecco's Medium (IMDM) + Glutamax (Gibco, Thermo Fischer Scientific) supplemented with 10% heat‐inactivated fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen) at a final concentration of 1 × 106 cells/ml and seeded (100 μl per well) in 96‐well tissue culture plates. PBMCs were exposed to peptides at final concentrations of 1, 10, and 100 μg/ml. Exposure to LPS (1 μg/ml) was used as a positive control, and cells with only IMDM served as negative control. After exposure for 24 hr, cells were incubated with Annexin V‐APC and propidium iodide (eBiosciences), and using flow cytometry (FACS Canto II, BD Biosciences), the proportions of live (unstained), dead (PI only), early‐apoptotic (Annexin V only), and late‐apoptotic (Annexin V + PI) cells were determined (BD FACSDiva). Data are presented as mean values ±SD.
Annexin 5 apc and propidium iodide
Manufactured by Thermo Fisher Scientific
Sourced in United States
Annexin V‐APC and propidium iodide are laboratory reagents used for the detection and analysis of apoptosis (programmed cell death) in cell samples. Annexin V‐APC binds to phosphatidylserine, a molecule that is exposed on the surface of apoptotic cells. Propidium iodide is a DNA‐binding dye that can enter cells with compromised membranes, such as late‐stage apoptotic or necrotic cells. Together, these two reagents allow for the identification and quantification of different stages of cell death within a sample.
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Lab products found in correlation
2 protocols using annexin 5 apc and propidium iodide
1
Isolation and Characterization of Human PBMC Monocytes
2
Assessing Apoptosis in Neurospheres
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Dissociated cells from neurospheres previously grown and treated in 6 well plates were stained with Annexin V-APC and propidium iodide (eBioscience, Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s protocol, utilizing Annexin V staining buffer. Neurospheres from one well of six well plates were treated as one replicate for each condition tested. A total of 5 to 6 independent replicates were performed per condition tested. Samples stained with Annexin V-APC + PI, as well as unstained and single-stained controls, were analyzed using a BD Canto Flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Compensation parameters were established using the BD DIVA acquisition software prior to the acquisition of stained samples. The proportion of apoptotic cell death (Annexin V+ and PI+ or PI− cells) and non-apoptotic cell death (PI+ and AnnexinV-cells) was quantified from all singlet cells using FlowJo (v10.9), after fluorescence compensation and gating based on single-color stained samples.
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