Apoptosis of MDSCs was measured using the APC Annexin V apoptosis kit (Biolegend). In brief, MDSCs for all experiments were generated and treated in vitro or isolated from mice. The cells were stained in FACS buffer (1% FCS in PBS) for surface MDSC markers (CD11b, Ly6C and Ly6G) and Aqua LIVE/DEAD Fixable Dead Cell Stain for 30 min at RT. Cells were washed with 1 ml of PBS and twice with 1X Binding Buffer (Biolegend) and resuspended in 200 µl of Binding Buffer. In each tube 5 µl of APC Annexin V was added and incubated for 15 min at RT. For the final step, 400 µl of Binding Buffer was added, and the cells were immediately analyzed using the BD LSRFortessa™ Cell Analyzer (BD Bioscience) and analysis was performed using FlowJo V10 software. Apoptosis rate was calculated based on the frequency of Aqua+Annexin V+ in each population (M-MDSC: CD11b+ Ly-6C+ Ly-6G- or PMN-MDSC: CD11b+ Ly-6C- Ly6G+) cells relative to untreated controls.
Apc annexin 5 apoptosis kit
Manufactured by BioLegend
The APC Annexin V apoptosis kit is a laboratory reagent used to detect and measure apoptosis, a form of programmed cell death, in cell samples. The kit utilizes Annexin V, a calcium-dependent phospholipid-binding protein, conjugated to the fluorescent dye allophycocyanin (APC) to identify cells undergoing apoptosis.
Automatically generated - may contain errors
Lab products found in correlation
Binding buffer, by BioLegend (1 mentions)
Lsrfortessa cell analyzer, by BD (1 mentions)
Antimycin a, by Merck Group (1 mentions)
Rotenone, by Merck Group (1 mentions)
Cd11b m1 70, by BioLegend (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Anti cd16 cd32 clone 93, by BioLegend (1 mentions)
Facsaria 3 sorter, by BD (1 mentions)
3 protocols using apc annexin 5 apoptosis kit
1
Quantifying MDSC Apoptosis by Flow Cytometry
2
Murine MDSC Differentiation and Functional Assays
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Bone marrow (BM) cells were harvested from the femur and tibia of the mice and red blood cells (RBCs) were removed using RBC lysis buffer. A total of 2 × 106 BM cells were cultured in 3 ml RPMI supplemented with 10% FBS, 1% L-glutamine, and 1% Penicillin/Streptomycin, in addition to granulocyte-macrophage colony-stimulating factor (rmGM-CSF) (40 ng/mL) and interleukin-6 (rmIL-6) (40 ng/mL) for 4 days. On day three, half of the media was gently replaced with fresh differentiation media. MDSCs (purity > 95% CD11b + ) were harvested on day 4 for subsequent experiments. For isoproterenol (ISO) treatment, 10 µM ISO was added to WT cell culture media starting day 0 and added fresh daily. Similarly, β2-AR-/- MDSC controls were generated with and without ISO treatment. To examine cell viability post electron chain transport (ECT) blockade in MDSCs, increasing concentrations of metformin hydrochloride: 0−5 mM (Cayman BioSystems), rotenone: 0−10 µM (Sigma), and antimycin A: 0−1 µM (Sigma) were added on day 3 of differentiation. Apoptosis was measured by the APC Annexin V apoptosis kit (Biolegends) as described below.
3
Comprehensive Immune Cell Profiling
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Antibodies against mouse CD45 (30-F11), TCRB (H57-597), H-2Kb (AF6-88.5), CD3 (17A2), CD19 (6D5 or 1D3), NK1.1 (PD136), Ly6C (HK1.4), F4/80 (BM8), CD11c (N418), CD8a (53–6.7), and CD11b (M1/70) were from BioLegend. Antibodies against mouse MHCII (M5/114.15.12) were from BD Biosciences and antibodies against CD103 (M290) were from BD Horizon. Prior to staining, cells were washed with FACS staining buffer (chilled PBS containing 2% FBS). For cell sorting, 2 mM EDTA (Thermo Fisher Scientific) was added to FACS staining buffer. Cells were stained for 15 min on ice with eBioscience Fixable Viability Dye eFluor 780 to distinguish live and dead cells and with anti-CD16/CD32 (clone 93, BioLegend) to prevent non-specific antibody binding. Cells were then washed once and cell surface proteins were stained for 30 min on ice with fluorophore-conjugated antibodies. Annexin-V and propidium iodide (PI) staining was performed using the APC Annexin-V Apoptosis Kit (BioLegend). Flow cytometry sample acquisition was performed on a BD FACS Symphony A3 cytometer, and the collected data were analyzed using FlowJo V.10.5.3 software (TreeStar). For tumor cell sorting, cells were harvested from cultures and sorted into DMEM growth medium using a BD FACSAria III sorter.
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