8–10 weeks old male mice were injected i.p. with 250 uL PBS containing 250 mg dexamethasone (Sigma-Aldrich) dissolved in DMSO. Eighteen hours after injection, the mice were euthanized, and thymi were harvested and weighed. One lobe of the thymus was mechanically disaggregated, and cells number was determined using Countess II (Invitrogen). 1×106 cells were stained with either FITC-conjugated Annexin V antibody (Biolegend) or anti-F4/80 antibody (Biolegend) to determine apoptotic cells and macrophage number, respectively. The other thymus lobe was formalin-fixed, paraffin-embedded, and sectioned, followed by immunostaining the sections with TUNEL reagent (Roche), anti-TGFβ1 (Abcam), anti-phospho-ERK1/2 (Cell Signaling), anti-ERK1/2 (Cell Signaling), anti-DUSP4 (Abcam), and/or anti-Mac2 antibodies (Cedarlane). In situ efferocytosis was quantified by counting TUNEL+ cells that were associated with Mac2+ cells versus macrophage-free TUNEL+ cells. Macrophage-associated apoptotic cells followed the criteria of TUNEL+ nuclei surrounded by or in contact with neighboring Mac2+ macrophages. Free apoptotic cells exhibited nuclear condensation and were not in contact with neighboring macrophages.
Fitc conjugated annexin 5 antibody
Manufactured by BioLegend
Sourced in United States
FITC-conjugated Annexin V is a protein that binds to phosphatidylserine, a cell surface marker of apoptosis. It is used in flow cytometry and fluorescence microscopy applications to detect and quantify apoptotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti dusp4, by Abcam (2 mentions)
Tunel reagent, by Roche (2 mentions)
Anti f4 80 antibody, by BioLegend (2 mentions)
Anti mac2 antibody, by Cedarlane (2 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Countess 2, by Thermo Fisher Scientific (2 mentions)
Anti erk1 2, by Cell Signaling Technology (2 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (2 mentions)
Anti tgf β1, by Abcam (2 mentions)
Pi conjugated antibody, by BioLegend (2 mentions)
Facscanto 2, by BD (1 mentions)
7 amino actomycin d, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Annexin 5 binding buffer, by BioLegend (1 mentions)
6 protocols using fitc conjugated annexin 5 antibody
1
Dexamethasone-Induced Thymus Apoptosis in Mice
2
Dexamethasone-Induced Thymus Apoptosis in Mice
3
Cell Cycle and Apoptosis Analysis
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Cells were seeded and allowed to adhere overnight before treated with 1 µM AZD6738. After 96 h of treatment, cells were collected, washed, and stained with propidium iodide (PI) (0.1% sodium citrate, 0.1% Triton X-100, and 50 µg/ml PI) as described previously48 (link). For analysis of apoptosis, cells were collected at various time points after treatment with 1 µM AZD6738, washed with cold Hank’s Balanced Salt Solution, and stained for 20 min in the dark at room temperature with 25 µl/ml of a FITC-conjugated annexin V-antibody from Biolegend (San Diego, CA, USA). Directly before flow cytometric measurement, 10 µg/ml PI was added to the staining solution. Cell cycle distribution and apoptotic cells were quantified by using the BD FACSCanto II from BD Biosciences (San Jose, CA) and the FlowJo v10 software from FlowJo, LLC (Ashland, OR). At least 20,000 gated events per sample were analyzed.
4
Cell Cycle Dynamics and Apoptosis upon LINC01133 Knockdown
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For FACS based analysis of the cell cycle changes upon LINC01133 knockdown, we used standard propidium iodide (PI) DNA staining protocol. In brief, transfected cells were harvested 72 h after siRNA knockdown and 1 × 106 cells were fixed in 70% precooled ethanol for 2 h on ice. After washing with PBS (Thermo Fisher Scientific, Waltham, MA, USA) the cells were re-suspended in 0.5 mL PI/RNAse containing staining buffer (550825), BD Pharminogen™, Heidelberg, Germany) supplemented with 10 μL PI staining solution (51-6621-1E, BD Pharmingen™, Heidelberg, Germany). After incubation for 15 minutes at room temperature, the number of PI positive cells was measured by flow cytometry. The effect of LINC01133 on cellular apoptotic rate was evaluated using staining with AnnexinV (FITC-conjugated antibody (640906), BioLegend, San Diego, CA, USA; Annexin V Binding Buffer, (422201), BioLegend, San Diego, CA, USA) in conjunction with the vital dye 7-amino-actomycin D (00-6993-50) eBioscience, San Diego, CA, USA) followed by flow cytometry measurement. A total of 1 × 106 cells 72 h after transfection was used in the analysis, with a total of 10,000 events recorded for each sample, and unstained cells being used as assay control.
5
RA Fibroblast Apoptosis Assay
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RA synovial fibroblast cells (Patient No. R41 to No. R45, n = 5) were cultured and treated with or without MRS2578 at a final concentration of 10 μM for 1 h. Then, UDP at a final concentration of 100 μM was added, and incubation was continued for 24 h. Cells (6 × 104) were collected and resuspended in binding buffer. An Annexin V-FITC-conjugated antibody and a PI-conjugated antibody (BioLegend) were added to the suspended cells. Apoptosis was detected by flow cytometry.
6
Apoptosis in Synovial Fibroblasts
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RA synovial broblast cells (Patient No. R41 to No. R45, n = 5) were cultured and treated with or without MRS2578 at a nal concentration of 10 μM for 1 h. Then, UDP at a nal concentration of 100 μM was added, and incubation was continued for 24 h. Cells (6×10 4 ) were collected and resuspended in binding buffer. An Annexin V-FITC-conjugated antibody and a PI-conjugated antibody (BioLegend) were added to the suspended cells. Apoptosis was detected by ow cytometry.
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