Cells were treated with PD-325901 and/or GDC-0941 at 100 nM and 1 μM concentration respectively for 24 h or 48 h. Floating cells were centrifuged at 1,000 g for 10′. Attached cells were washed with PBS and trypsinized before re-suspending them in the appropriate media. Cells suspensions were centrifuged at 300 g for 10′, re-suspended in cold PBS before a second round of centrifugation at 300 g for 10′. The fractions of floating cells and attached cells were pooled in 1X annexinV binding buffer and re-suspended. Cells were counted and 100 μl of cell suspension containing 105 cells was transferred to a 5 ml culture tubes. BD Pharmingen FITC annexinV Apoptosis Detection Kit I (BD Biosciences) was used for the staining. 5 μl of FITC annexinV and 10 μl of PI were added to the suspension, gently vortexed and incubated for 15′ at room temperature in the dark before measuring by flow cytometry within 1 h.
Pharmingen fitc annexin 5 apoptosis detection kit 1
Manufactured by BD
Sourced in United States
The BD Pharmingen™ FITC Annexin V Apoptosis Detection Kit is a laboratory tool used to detect and analyze apoptosis, a process of programmed cell death. The kit utilizes FITC-conjugated Annexin V, a protein that binds to a cellular component called phosphatidylserine, which is exposed during apoptosis. This allows for the identification and quantification of apoptotic cells.
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Lab products found in correlation
Facscalibur, by BD (4 mentions)
Facscalibur flow cytometer, by BD (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Facscanto 2, by BD (2 mentions)
Lsrfortessa, by BD (2 mentions)
Cytoflex s flow cytometer, by Beckman Coulter (2 mentions)
Facscalibur cytometer, by BD (2 mentions)
Facscalibur flow cytometer, by Beckman Coulter (2 mentions)
Epics xl mcl flow cytometry, by Beckman Coulter (2 mentions)
Onestep rt pcr kit, by Qiagen (1 mentions)
Gibco dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
Cisplatin cddp, by Merck Group (1 mentions)
Sulforhodamine b, by Merck Group (1 mentions)
Tris base, by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Acetic acid, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Bricyte flow cytometer, by Mindray (1 mentions)
Ros assay kit, by Beyotime (1 mentions)
45 protocols using pharmingen fitc annexin 5 apoptosis detection kit 1
1
Apoptosis Induction by Kinase Inhibitors
2
Quantifying Cell Apoptosis by Flow Cytometry
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The trypsinized cells were collected and cell apoptosis was determined using BD Pharmingen™ FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, USA) according to the manufacturer’s instructions. The cells were washed twice with cold PBS and then resuspended in 500 μl 1 × binding buffer to a concentration of 1 × 106 cells/mL. After adding 5 μl FITC Annexin V and 5 μl propidium iodide (PI), the cells were gently vortex and incubated at room temperature in the dark for 15 min and finally analyzed by a flow cytometer (Beckman Coulter, Fullerton, CA, USA).
3
Quantification of Apoptosis in Cancer Cells
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For analysis of apoptosis, the BD Pharmingen FITC Annexin V Apoptosis Detection Kit I (BD Biosciences) was used according to the manufacturer’s instructions. Briefly, MDA-MB-231, MDA-MB-453 and HCC1937 cells were seeded into 6-well plates. The following day, cells were incubated with atuveciclib (0.5 or 1 or 3 µM) for four days. Following treatment, cells were harvested using trypsin, washed three times with PBS, and stained with Propidium Iodide Staining Solution (PI) and FITC Annexin V. Stained samples were analyzed by flow cytometry (BD LSRFortessa Analyzer cytometer) and FlowJo software (version 10.3). Percentage of apoptotic cells (Annexin-V positive and Annexin-V+PI double positive) was quantified.
4
Annexin V Apoptosis Assay
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Percentage of apoptotic cells was measured using BD Pharmingen FITC AnnexinV apoptosis detection kit I (556547; Erembodegem, Belgium) according to the manufacturers' protocol. Hoechst 33258 (H3569), (Molecular probe/Invitrogen, Leiden, the Netherlands) was used at the concentration of 1 µg/ml. Briefly, harvested cells (1×10 ∧5) were washed with PBS, centrifuged and resuspended in Annexin buffer provided with the kit. The final concentration used for Annexin was 0.5 µg/µl. Cells were analyzed by FACSAria III machine.
5
Evaluating Cisplatin Cytotoxicity and Apoptosis
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The compound cisplatin (CDDP, #479306; ≥99.9% purity) was purchased from Sigma Aldrich Co. (St. Louis, MO, USA). Stock solution of 1 mM dissolved in dimethyl sulfoxide (DMSO, #W387520; Sigma-Aldrich Co., St. Louis, MO, USA) was stored at −20 °C until use. CDDP was dissolved in DMSO and diluted further to desired concentration in sterile complete culture medium immediately prior to use. Propidium iodide (PI, #81845), phosphate buffered saline (PBS, #P7059), sulforhodamine B (SRB, #230162) dye, TRIS base (#93352), and acetic acid (#A6283) were all purchased from Sigma (Sigma-Aldrich Co., St. Louis, MO, USA), Gibco® Dulbecco’s modified Eagle medium (DMEM, #11960077), fetal bovine serum (FBS, #A31605), and trypsin-EDTA (#25300054) were purchased from Thermo Fisher (Thermo Fisher Scientific Inc., Waltham, MA, USA). The QIAGEN OneStep RT-PCR kit (#210212) was obtained from Qiagen (QIAGEN Inc., Germantown, MD, USA). BD Pharmingen™ FITC Annexin-V apoptosis detection kit I (#556547) was from BD (BD Biosciences, San Jose, CA, USA).
6
Apoptosis Evaluation by Flow Cytometry
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A FACSCalibur Flow Cytometer (BD Biosciences, USA) was used for the apoptosis assay followed the product manual. Cells were seeded in 6-well plates, harvesting in centrifuge tube after different treatment. Then washing, resuspension and staining were in accordance with the BD Pharmingen™ FITC Annexin V Apoptosis Detection Kit I (BD) protocol. The data were then analyzed with Flow Jo 10.0 software (Tree Star, San Francisco, CA, USA). Each assay was independently repeated three times.
7
Apoptosis and Autophagy in HUVECs
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At 48 h after transfection, HUVECs were trypsinized, collected and washed with pre-cooled PBS twice. The apoptosis of cells was examined by flow cytometry using BD Pharmingen FITC Annexin V Apoptosis Detection kit I (cat. no. 556547; BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's protocol. Early apoptotic cells were identified by positive staining with Annexin V only, necrotic cells were indicated by positive staining with propidium iodide only, and late apoptotic cells were identified by double-positive staining with propidium iodide and Annexin V. In order to observe whether autophagy regulates apoptosis in HUVECs, the cells were incubated with autophagy inhibitor 3-methyladenine (3-MA; 50 nM) for 24 h prior to flow cytometric analysis.
8
Quantifying Apoptosis in MDA-MB-231 Cells
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Apoptosis was measured using the BD Pharmingen™ FITC Annexin V Apoptosis Detection Kit I (cat. 556547, BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. MDA-MB-231 cells were seeded (2.5 × 105 cells/well) in 35 mm culture dishes. The day next, the cells were either treated with erucin (30 μM) for 48 h or left untreated. MDA-MB-231 cells were later collected and stained with Annexin V-FITC/Propidium Iodide (PI). Flow cytometry analysis was performed using a BriCyte flow cytometer (Mindray, Italy). A minimum of 50,000 events for each sample were collected and data were analyzed using FlowJo v10 software (Tree Star, Ashland, OR USA).
9
Apoptosis Assay of Neurospheres
10
Osteoclast Apoptosis and Mitochondrial Dynamics
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BMMs were cultured in M-CSF and RANKL containing medium with or without P.M OMVs for 5 days, and a BD Pharmingen™ FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, San Jose) and a JC-1 mitochondrial membrane potential assay kit (Bestbio, Shanghai) were used to detect apoptosis and mitochondrial membrane potential (MMP) ΔΨm, respectively. For MMP measurement, osteoclasts were collected after treatment with P.M OMVs for 5 days, stained with JC-1 for 20 min at 37°C in dark, washed twice with 1 x FACS buffer, and analyzed by flow cytometry. ROS production was detected utilizing a ROS Assay Kit (Beyotime, Shanghai). Apoptosis, ROS, and MMP were detected and analyzed by the BD FACSCanto II (BD Biosciences) and Flow Jo 10 software (BD Biosciences), respectively. Gating strategy for FACS is given in Figure SF6
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