To sort CD4+ T cells from the spleen cell surface staining was performed in PBS, 1% BSA (Sigma), 1 mM EDTA (AppliChem). Sorting was performed on MoFlo Astrios. Annexin V binding buffer, fluorochrome-conjugated Annexin V and 7-AAD were purchased from Biolegend and used according to manufacturer’s instructions. Briefly, cells were washed once with PBS and once with Annexin V binding buffer. Cells were stained with Annexin V (1:200 dilution) for 15 min at 4 °C in Annexin V binding buffer. Data were acquired on an Accuri C6 (BD) flow cytometer and analysed in FlowJo. The induced cell death was calculated as [(% death with CMA treatment − % death without CMA treatment)/(100 − % death without CMA treatment)]. The FACS gating strategies are shown in Supplementary Fig. 10 .
Fluorochrome conjugated annexin 5
Manufactured by BioLegend
Sourced in United States
Fluorochrome-conjugated Annexin V is a protein that binds to phosphatidylserine, a phospholipid that is exposed on the surface of apoptotic cells. This protein is conjugated with a fluorescent dye, allowing for the detection and quantification of apoptotic cells using flow cytometry or fluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Annexin 5 binding buffer, by BioLegend (2 mentions)
7 aad, by BioLegend (1 mentions)
Ethylene diamine tetraacetic acid (edta), by AppliChem (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Accuri c6, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Facscanto, by BD (1 mentions)
Foxp3 buffer set, by BioLegend (1 mentions)
Fluorochrome conjugated antibodies, by BioLegend (1 mentions)
3 protocols using fluorochrome conjugated annexin 5
1
CD4+ T Cell Sorting and Apoptosis Assay
2
Annexin V-PI Apoptosis Assay by Flow Cytometry
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The NRFi-MDA cells were seeded in 60 cm2 dishes at a density of 1×105 cells/well and cultured overnight. After the Pba-laser irradiation, the cells were trypsinized and spun down at 15,000 rpm for 10 min at 4°C. Then, 2×105 cells were transferred to 1.5 ml tube for centrifugation at 8,000 g for 5 min at 4°C. Then, 5 µl of fluorochrome conjugated Annexin V (BioLegend, San Diego, CA, USA) and 10 µl of propidium iodide (PI, BioLegend) solution were added into each tube and incubated with gentle vortex. The cells were incubated for 15 min at room temperature in the dark and then, 400 ul of Annexin V binding buffer (BioLegend) was added to each tube. Stained cells were analyzed using a Becton-Dickinson FACS Canto (SanJose, CA, USA) with and data were analyzed with FACSDiva software (BD).
3
Multi-Parameter Flow Cytometry Analysis
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Fluorochrome-conjugated antibodies against mouse CD3, CD4, CD8, CD19 and Ki-67, and against human Ki-67, Zombie-Green/Violet fixable viability dyes and Foxp3 buffer set were purchased from Biolegend (San Diego, CA). Lymphocytes were stained for cell surface markers.
Live and dead cells were distinguished by staining the cells with Zombie dyes or 7-AAD. For Ki-67 staining, cells were fixed with CytoFix/CytoPerm buffer, washed with PBS plus 1% FBS and 1x CytoPerm buffer, and incubated with antibodies against mouse or human Ki-67. Jurkat and Raji cells were stained with Zombie-Green dye followed by fixation, permealization and staining with anti-human Ki-67. (Biolegend, San Diego, CA). For Annexin V staining, cells were washed twice with plain PBS and once with Annexin V binging buffer (10mM HEPES, pH7.4, 140mM NaCl, 2.5mM CaCl 2 ) and incubated with fluorochrome-conjugated Annexin V (Biolegend) in Annexin V binding buffer for 15min at room temperature. The cells were washed twice in Annexin V binding buffer, and re-suspended in Annexin V binding buffer. 7-AAD was added to the cells before analyzed by flow cytometry to distinguish dead and live cells.
Live and dead cells were distinguished by staining the cells with Zombie dyes or 7-AAD. For Ki-67 staining, cells were fixed with CytoFix/CytoPerm buffer, washed with PBS plus 1% FBS and 1x CytoPerm buffer, and incubated with antibodies against mouse or human Ki-67. Jurkat and Raji cells were stained with Zombie-Green dye followed by fixation, permealization and staining with anti-human Ki-67. (Biolegend, San Diego, CA). For Annexin V staining, cells were washed twice with plain PBS and once with Annexin V binging buffer (10mM HEPES, pH7.4, 140mM NaCl, 2.5mM CaCl 2 ) and incubated with fluorochrome-conjugated Annexin V (Biolegend) in Annexin V binding buffer for 15min at room temperature. The cells were washed twice in Annexin V binding buffer, and re-suspended in Annexin V binding buffer. 7-AAD was added to the cells before analyzed by flow cytometry to distinguish dead and live cells.
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