Apoptosis detection was based on the Annexin-V molecule binding to phosphatidylserine membrane molecules. Two cell lines were used to study apoptosis: Ad-MSC 3 cells were plated in a 5 × 104 cells/well concentration, while 4 × 104/well A431-mCh were seeded into each well in a 12-well tissue culture plate. Cells were incubated overnight, and treated with cisplatin, doxorubicin, and nutlin-3 at the indicated concentrations in 1 mL of culture media. At day 5, cells were trypsinized and centrifuged, while the supernatant was also collected and combined with the detached cells to include all already dead cells. Cells were washed twice with 500 µL of 1% BSA-PBS (bovine serum albumin dissolved in PBS, A8022, Sigma-Aldrich, St. Louis, MO, USA) solution and centrifuged at 400× G for 4 min. Cells were resuspended in 100 µL of 1× Annexin Binding Buffer (422201, Biolegend, San Diego, CA, USA), and 3 µL of Annexin V-Pacific Blue (A35122, Biolegend, San Diego, CA, USA) and 1 µL of TO-PRO-3 (R37170, Invitrogen, Waltham, MA, USA) were added to each sample. After 15 min of incubation, labeled and non-labeled samples were diluted 5-fold with 1× Annexin Binding Buffer and analyzed by Attune™ NxT flow cytometer.
Annexin binding buffer
Manufactured by BioLegend
Sourced in United States
Annexin binding buffer is a reagent used in flow cytometry and other research applications to facilitate the binding of Annexin V to phosphatidylserine on the surface of cells. It provides the necessary ionic conditions and pH for the interaction between Annexin V and phosphatidylserine.
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Lab products found in correlation
Annexin 5 fitc, by BioLegend (4 mentions)
Annexin 5 apc, by BioLegend (3 mentions)
Lsrfortessa, by BD (2 mentions)
Annexin 5 pacific blue, by BioLegend (1 mentions)
A8022, by Merck Group (1 mentions)
Ultracomp ebeads, by Thermo Fisher Scientific (1 mentions)
True nuclear transcription factor buffer set, by BioLegend (1 mentions)
Trustain fcx anti mouse cd16 32 antibody, by BioLegend (1 mentions)
Annexin binding buffer, by Beckman Coulter (1 mentions)
Facsverse flow cytometer, by BD (1 mentions)
Staurosporine, by Santa Cruz Biotechnology (1 mentions)
Fitc conjugated annexin 5, by BioLegend (1 mentions)
7 aminoactinomycin d, by BioLegend (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Sytox green, by Thermo Fisher Scientific (1 mentions)
Fluo 4 am, by Thermo Fisher Scientific (1 mentions)
Fortessa, by BD (1 mentions)
Annexin 5 alexa fluor 647 conjugate, by Thermo Fisher Scientific (1 mentions)
Annexin 5 alexa fluor 647 conjugate, by BioLegend (1 mentions)
Facscalibur, by BD (1 mentions)
16 protocols using annexin binding buffer
1
Apoptosis Detection in Cell Lines
2
Multi-parameter Flow Cytometry for Cell Characterization
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For surface staining, cells were counted, washed with 1× phosphate-buffered saline (PBS) (pH 7.4) and non-specific sites were blocked by using TruStainfcX (anti-mouse CD16/32) antibody (BioLegend) for 5 min. Cells were stained with indicated antibodies for 20 min at room temperature (RT) in dark. For intracellular staining, cells were fixed in fixation buffer (200 µL/sample) for 20 min, washed twice in 1 mL of perm/wash buffer (5 min each) and indicated antibodies were added for 30 min at RT in dark. For nuclear staining, True-Nuclear Transcription Factor Buffer Set (BioLegend) was used. The cells were stained with Annexin-V and 7AAD in Annexin binding buffer (BioLegend) for cell death assays. The multi-color flow cytometry compensation was achieved using UltraComp eBeads (Thermo Fisher Scientific) and events were captured using flow cytometer (BD LSR Fortessa). The unstained and isotype control antibodies were used for gating. Data were analyzed by using FlowJo V.10 software (FlowJo, LLC).
3
Annexin V-PI Apoptosis Assay in mProx24 Cells
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The mProx24 cells were treated with CsA or CsA plus 5‐ALA/SFC and then were plated in six‐well plates. After incubation for 12 and 24 h, the cells were stained with FITC‐Annexin V (Biolegend, Tokyo, Japan) and propidium iodide (PI) and washed with PBS. They were then centrifuged at 270 g . for 5 min. The cells were resuspended at a density of 1 × 106 cells per mL with Annexin‐binding buffer (Biolegend). To every 100 μL of cell suspension, 5 μL of FITC‐Annexin V and 2 μL of PI were added. The cells were incubated at room temperature for 20 min. Then, 500 μL of 1× Annexin‐binding buffer was added, and the cells were analyzed immediately by flow cytometer (Gallios; Beckman Coulter, Tokyo, Japan).
4
Apoptosis Detection by Annexin V/PI
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Cells were trypsinized and collected. The cell pellet was re-suspended in annexin-binding buffer (BioLegend, San Diego, CA, USA) and subsequently stained with annexin V-FITC (BioLegend) and PI for 10 min in the dark. The stained cells were examined using a FACSCalibur system.
5
Apoptosis Induction and Detection
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The cells were treated with DMSO or idasanutlin for 48 h, or with Staurosporine (Santa Cruz Biotechnology, Dallas, TX, USA), a well-known inducer of apoptosis, for 16 h. Following this treatment, the cells were trypsinized and collected together with growth media and PBS, which was used for washes, to avoid any loss of detached apoptotic cells. The cells were centrifuged, washed with PBS, centrifuged again, suspended with annexin-binding buffer (BioLegend) and stained with 5 µg/mL of FITC-conjugated annexin V (BioLegend) and 2.5 µg/mL 7-Aminoactinomycin D (7-AAD, BioLegend) for 15 min at room temperature. The samples were analyzed using BD FACSVerse flow cytometer (Becton Dickinson) with appropriate color compensation.
6
Quantifying Apoptosis in Cell Lines
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All cell lines were trypsinized, plated in a 6-well plate, and allowed to attach overnight. Cells were then treated with the indicated treatments and both floating and adherent cells were harvested 48 hours after the start of treatment. Cells were then incubated with SYTOX Green (Cat #S7020; Invitrogen/Thermo Fisher Scientific) and allophycocyanin-conjugated annexin V (APC Annexin V; Cat #640941; BioLegend, San Diego, CA, USA) for at least 20 minutes in Annexin Binding Buffer (BioLegend) according to the manufacturer's instructions. A FACSCanto II Flow Cytometer (Cat #338960; BD Biosciences, San Jose, CA, USA) was then used to analyze the samples; a total of 10,000 events were collected for each sample. The percentage of annexin-positive cells was determined using FlowJo software (version 10.4.2; FlowJo, Ashland, OR, USA).
7
Measurement of Calcium Signaling and Apoptosis
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Cells were treated with ionomycin for 15 min at 37 °C in Ringer solution containing 2 mM CaCl2. Cells were stained for 30 min on ice and flow cytometry was performed using a BD Fortessa (BD Bioscience). Intracellular Ca2+ concentration was monitored with 1 μM Fluo-4 AM (Life Technologies) loaded in cells for 30 min at 37 °C prior to the experiment. Surface exposure of phosphatidylserine was detected using 9 ng/ml Annexin V staining in Annexin binding buffer (BioLegend). Microvesicles were distinguished from apoptotic bodies using the fixable viability dye eFluor780 dye (eBioscience). Surface staining for receptors was carried out for 30 min on ice using the following antibodies: PD-1 APC (MIH4), ICAM-1 PE (HA58), Transferrin receptor (TfR) PE (M-A712) from BD Biosciences and CD28 PE (CD28.2), LFA-1 PE (HI111), CD4 PE (OKT4), CD3 PE/Cy7 (UCHT1), HLA ABC PE (W6/32) from BioLegend. Data were analyzed using FlowJo Software (Treestar).
8
Evaluation of CD19 Expression in P493-6 Cells
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We performed surface staining to detect CD19 expression using an N-terminal–specific antibody in P493-6 cells treated with doxycycline (0.1 μg/ml) for 24 h. Briefly, one million P493-6 cells per replicate were harvested, washed twice with 1× PBS, and stained with rabbit anti–human CD19 polyclonal antibody (MBS9204578; MyBioSource, LLC; 1:500 dilutions) for 10 min at room temperature followed by goat anti–rabbit Alexa Flour 594 secondary antibody (A11012; Life Technologies; 1:1,000 dilutions) incubation for 10 min at room temperature. Staining was done in 10% FBS in 1× PBS. For CD3 staining we used mouse anti–CD3-FITC antibody (555339; BD Pharmingen), and for CD20 staining we used mouse anti–CD20-PE/Cy5 antibody (302308; BioLegend). To detect apoptotic cell death, we used Annexin V Alexa Fluor 647 conjugate (A23204; Invitrogen). Live cells were stained with CD3-FITC antibody, CD20-PE/Cy5 antibody, and Annexin V Alexa Fluor 647 conjugate in Annexin binding buffer (79998; BioLegend) for 20 min at room temperature. Flow cytometry detection of CD19 staining was performed using the Guava easyCyte instrument. Data were analyzed using FlowJo software. We used two experimental replicates and performed experiments in three biological replicates. Statistical significance was calculated using a two-tailed Student’s t test.
9
Apoptosis detection in THP-1 cells
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After staining THP-1 cells with fluorescence-conjugated antibodies and Live/Dead fixable marker as described above, the cells were washed and re-suspended in Annexin-binding buffer (BioLegend) containing Annexin V FITC (BioLegend) for 20 minutes AT RT. After another round of washing the cells were re-suspended in Annexin-binding buffer and analysed by flow cytometric analysis. The absolute number of human cells was analysed by a high-throughput automated plate reader (BD LSRFortessa).
10
Annexin V and PI Apoptosis Assay
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For analyzing apoptosis cells were labeled with FITC Annexin V (BioLegend, San Diego/USA) and Propidiumiodide (PI) (InvitrogenTM by life technologies TM AG (Carlsbad/USA)) supernatant was harvested to collect possible apoptotic cells. After that, cells were washed with PBS, trypsinized and dissolved with the collected supernatant; 2 × 105cells were used. The cells were transferred to a 15 ml Falcon tube and centrifuged 3 min at RT and 2000 × g. The supernatant was removed and the pellet was washed twice, first with PBS, secondly with Annexin-Binding Buffer (BioLegend(San Diego, USA)). Subsequently, the pellet was resuspended in 100 μl Annexin-Binding Buffer and cells were incubated with 5 μl of Annexin V, and 2 μl of PI 1 mg/ml for 15 min at RT in the dark. Finally, 100 μl Annexin-Binding Buffer was added. For positive control 1 μM Staurosporine (Sigma Aldrich Co. LLC, St. Louis/USA) was added for 4 h to HaCaT cells to induce apoptosis. The subsequent measurement was performed on a BD FACSCalibur (BD Biosciences, Heidelberg, Germany) while Flowing Software version 2.5.1 was used to perform a distribution analysis for statistical evaluation. Experiments were conducted in triplicates and repeated twice.
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