Apoptosis was determined by two‐color analysis using propidium iodide (PI) and FITC‐conjugated anti‐Annexin V (BD Pharmingen, USA) according to the manufacturer's instructions. Cells were harvested and washed three times with PBS then cells were stained with PI and FITC‐conjugated anti‐Annexin V and analyzed with a FACSCalibur flow cytometer.
Fitc conjugated anti annexin 5
Manufactured by BD
Sourced in United States
FITC-conjugated anti-Annexin V is a laboratory reagent used for the detection and analysis of Annexin V, a calcium-dependent phospholipid-binding protein. This conjugated antibody can be used in flow cytometry and other immunoassays to identify and quantify Annexin V expression in cells.
Automatically generated - may contain errors
Lab products found in correlation
Pe conjugated anti ros, by Abcam (2 mentions)
Pe conjugated anti ki67, by BD (2 mentions)
Flow cytometry, by Beckman Coulter (2 mentions)
Pe conjugated anti cxcr3, by BD (1 mentions)
Percp cy5, by BD (1 mentions)
Pe cy7 conjugated anti nk1, by BD (1 mentions)
Alexa fluor 647 conjugated anti foxp3, by BD (1 mentions)
Apc cy7, by BD (1 mentions)
Fixation permeabilization solution kit, by BD (1 mentions)
Fixation and permeabilization solution kit, by BD (1 mentions)
5 protocols using fitc conjugated anti annexin 5
1
Apoptosis Assay by Flow Cytometry
2
Cell Cycle and Apoptosis Analysis
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Cell cycle was analyzed following cell staining with propidium iodide (PI). Apoptosis was determined using propidium iodide (PI) and FITC-conjugated anti-Annexin V (BD Pharmingen). Cells were stained with PI and FITC-conjugated anti-Annexin V and analyzed with a FACScalibur flow cytometer.
3
Multicolor flow cytometry analysis
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This study used the following antibodies for flow cytometry (all from BD): FITC-conjugated anti–annexin V, anti-GL7, and anti-CD49d; PE-conjugated anti-CXCR3, anti-Fas, and anti-CD25; PerCP-Cy5.5–conjugated anti-B220 and anti-CD8; PE-Cy7–conjugated anti-NK1.1, anti-CD11a, and anti-CD45.1; allophycocyanin (APC)-conjugated anti-CD3; and APC-Cy7–conjugated anti-CD4 and anti-CD19. Alexa Fluor 647–conjugated anti-Foxp3 (BD) and PE-conjugated anti-Helios (BioLegend) antibodies were used for intracellular staining of T reg cells. Data were collected using a flow cytometer (LSR II; BD) and analyzed using FlowJo 7.6 software. To deplete NK1.1+ cells, mice were intraperitoneally injected with 1 mg anti-NK1.1 (clone PK136; purified from HB-191 culture supernatants), and persistent NK1.1+ cell depletion for at least 1 mo was confirmed by flow cytometry. For in vivo CXCR3, CTLA-4, or PD-L1 blockade, mice were injected intraperitoneally with 200 µg of functional anti-CXCR3 (CXCR3-173; BioLegend), CTLA-4 (9H10; Bio X cell), or PD-L1 (10F.9G2; BioLegend) every other day.
4
Quantifying Sertoli Cell Markers
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Sertoli cells were harvested to detect Ki67, Annexin V, and ROS, and the testes from the three groups were digested to generate single cells using 0.25% trypsin-EDTA. A Fixation/Permeabilization Solution Kit (BD, USA) was applied to fix and permeabilize the above cells. PE-conjugated anti-Ki67 (BD, USA), FITC-conjugated anti-Annexin V (BD, USA), PE-conjugated anti-ROS (Abcam, USA), and their corresponding isotypes were labeled for 30 min at 4 °C. Then, flow cytometry (Beckman, USA) was employed for analysis according to the manufacturer’s instructions.
5
Flow Cytometry Analysis of Sertoli Cell Markers
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To detect ROS, Ki67, and Annexin V, Sertoli cells were collected. The mouse testes were digested with 0.25% trypsin-EDTA to produce a single-cell suspension. Furthermore, the Fixation and Permeabilization Solution Kit (BD, USA) was used to fix and permeabilize the digested cells. The cells were labeled with FITC-conjugated anti-Annexin V (BD, USA), PE-conjugated anti-Ki67 (BD, USA), and PE-conjugated anti-ROS (Abcam, USA) antibodies and their isotype controls at 4 °C for 30 min. After that, flow cytometry (Beckman, USA) was used for analysis according to the manufacturer’s instructions.
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