PBS- and EV-treated K562 cells were lysed in RIPA buffer (Pierce) supplemented with a protease inhibitor cocktail (ThermoFisher Scientific). Cell and EV lysates were analyzed by SDS-PAGE and transferred onto PVDF (Bio-Rad). Membranes were blocked using 3% BSA and probed with specific antibodies. Proteins were detected using either Clarity (Bio-Rad) or WesternBright (Advansta) western ECL blotting substrates via chemiluminescence (ChemiDoc XRS+, Bio-Rad). Protein loading was normalized using anti-β-actin antibody (A5441, Sigma). Antibodies used for this study include: Alix (#2171), annexin V (#8555), Bcl-2 (#2872), CD9 (#13174), cleaved caspases -3 (#9664), -7 (#8438), -8 (#9496), and -9 (#9505), granzyme A (#4928), granzyme B (#4275), HSP70 (#4876), HSP90β (#5087), ICAM1 (#4915), LAMP-1 (#3243), phospho-STAT1: Ser727 (#9177) and Tyr701 (#9167), STAT1 (#9172), and VCAM1 (#13662) - (Cell Signaling); cytochrome c (sc-13156), full-length caspases -3 (sc-7272) and -7 (sc-28295), granulysin (sc-271119), MHC-1 (sc-55582), perforin-1 (sc-136994), Tsg101 (sc-136111), DNAM-1 (sc-376736) - (Santa Cruz); CD63 (SBI); rabbit polyclonal anti-LAMP-1 tail antibody; mouse monoclonal NKLAM and MHC-II antibodies made in-house. Horseradish peroxidase-labeled secondary anti-mouse and anti-rabbit antibodies were from Cell Signaling.
Annexin 5
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
Annexin V is a calcium-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine. It is commonly used in apoptosis assays to detect and quantify cells undergoing programmed cell death.
Automatically generated - may contain errors
Lab products found in correlation
Cd54 1 cam, by Cell Signaling Technology (2 mentions)
β tubulin, by Cell Signaling Technology (2 mentions)
Epcam, by Cell Signaling Technology (2 mentions)
Polyvinylidene fluoride (pvdf), by Bio-Rad (1 mentions)
Icam 1, by Cell Signaling Technology (1 mentions)
Vcam 1, by Cell Signaling Technology (1 mentions)
Lamp1, by Cell Signaling Technology (1 mentions)
Granzyme a, by Cell Signaling Technology (1 mentions)
Hsp90β, by Cell Signaling Technology (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Phospho stat1, by Cell Signaling Technology (1 mentions)
Propidium iodide, by Cell Signaling Technology (1 mentions)
Thiazolyl blue tetrazolium bromide mtt, by GoldBio (1 mentions)
Control sirna, by Horizon Discovery (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
Recombinant murine trail, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
13 protocols using annexin 5
1
Immunoblotting analysis of K562 cells and EVs
2
Comparative Analysis of PIK3CA Mutant and Wild-type Cell Lines
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T47D (PIK3CA mutant), HCC1500 (PIK3CA wild type and PTEN intact), and ZR75-1 (PIK3CA wild type and PTEN loss) cell lines were obtained from Dr. Dan R. Robinson’s laboratory. (University of Michigan Medical School, Ann Arbor, Michigan,United States). The MCF-7 (PIK3CA mutant) cell line was a kind gift from Dr. Jose Baselga’s laboratory (Memorial Sloan Kettering Cancer Center, New York, NY). We did not further test or authenticate the cells. The cells were cultured according to the manufacturer’s manual. Annexin V and propidium iodide (PI) were purchased from Cell Signaling Technology (Danvers, MA, United States). Moreover, 4-hydroxy-tamoxifen (tamoxifen) was purchased from Sigma Aldrich (St. Louis, MO, United States). Buparlisib and alpelisib compounds for in vitro and in vivo studies were supplied by Novartis (Basel, Switzerland). Thiazolyl blue tetrazolium bromide (MTT) was purchased from Gold Biotechnology (St Louis, MO, United States). siRNAs (siRNA-control, siRNA-PIK3CA, siRNA-PIK3CB) were purchased from GE Dharmacon (Lafayette, CO, United States) and used according to manufacturer’s instructions.
3
Analyzing Extracellular Vesicle Proteins
4
Quantifying TRAIL-Induced Apoptosis in Cells
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MS1 cells from the companion plates of the co-culture experiment were trypsinized and harvested at a concentration of 106 cells/250 µl. Cells were then stained with annexin V and PI (cat no. 6592; Cell Signaling Technology) as per manufacturer’s instructions. Percentage of cells undergoing early apoptosis was analyzed using a flow cytometer (LSR Fortessa; BD).
To analyze TRAIL-induced apoptosis, both endothelial cells (MS1) and differentiated trophoblast cells on day 5 of differentiation were treated with recombinant murine TRAIL (Peprotech) at a concentration of 6, 12, and 25 ng for 24 h at 37°C humidified incubator followed by annexin V and PI staining. Percentage of cells undergoing early apoptosis was analyzed using a flow cytometer (LSR Fortessa; BD).
To analyze TRAIL-induced apoptosis, both endothelial cells (MS1) and differentiated trophoblast cells on day 5 of differentiation were treated with recombinant murine TRAIL (Peprotech) at a concentration of 6, 12, and 25 ng for 24 h at 37°C humidified incubator followed by annexin V and PI staining. Percentage of cells undergoing early apoptosis was analyzed using a flow cytometer (LSR Fortessa; BD).
5
Investigating TIL Activation and Apoptosis
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CD32+ or CD32+ HVEM+ L cells were harvested, pulsed with 200 ng/mL OKT3 and co-cultured at a 1:1 ratio with TIL. After 12 h, TIL were harvested and stained for CD8 and intracellular IκBα PerCP-Cy5.5 (BD). After 5 days, TIL were stained for CD8, 7-AAD, Annexin-V, Bcl-xL Alexa 488 (Cell Signaling), Bcl-2 FITC (BD), and Bcl-6 PerCP-Cy5.5 (BD). Additionally, sorted CD8+ BTLA+ and BTLA− TILs were stimulated with 30 ng/mL plate-bound OKT3 and 10 μg/mL control human Ig, BTLA-Fc (R&D), or HVEM-Fc (R&D) with or without 10 μg/mL soluble anti-BTLA blocking mAb (clone 3B1) or PI3K inhibitor (10 nM GSK2126458). Cells were stained as described above.
6
Multimarker Immunostaining Protocol
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The following primary antibodies were used for staining: O4 (mouse hybridoma; Sigma-Aldrich, O7139, 1:100); PLP1 (Abcam, 28486, 1:1000); MOG (mouse monoclonal hybridoma supernatant, from B. Barres, Stanford University, USA, 1:50); MBP (AbD Serotec, MCA409S, 1:1000); STEM121 (Takara Bio, Y40410, 1:1000); AnnexinV (Cell Signaling Technology, 6592S, staining protocol provided by manufacturer); CD13 (BD Biosciences, 557454, 1:100). Secondary antibodies of appropriate species were purchased from Jackson ImmunoResearch (115165020) and Thermo Fisher Scientific (A-21245, A-21424, A-11006, A-21422). All secondary antibodies were used at 1:1000.
7
Quantitative Proteomics of Extracellular Vesicles
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Cells and exosomes were lysed in a lysis buffer (1% Triton-x100, 1 mM EDTA, 150 mM NaCl, 20 mM Tris pH 7.5) supplemented with 1 × cOmplete™ EDTA-free Protease Inhibitor Cocktail (11836170001, Roche (Basel, Switzerland)) and protein concentrations were determined using a Bradford assay (Bio-Rad) in relation to a BSA standard curve. Proteins were separated according to molecular weight by SDS-PAGE and transferred onto a PVDF membrane (Scientific Lab Supplies 10600023). For protein detection, membranes were blocked with 5% nonfat milk and 1% Tween/PBS, and subsequently incubated with primary antibodies against MMP-2 (Abcam, 86607, 1:500), EMMPRIN (Santa Cruz, 46700, 1:500), CD9 (Cell signalling, 13174, 1:1000), Alix (Cell signalling, 2171, 1:1000), Annexin V (Cell signalling, 8555, 1:1000) and Histone 4 (Cell signalling, 41328, 1:1000). Appropriate horseradish peroxidase-conjugated secondary antibodies were applied. GAPDH (Sigma-Aldrich, 406609, 1:2000) was used as the loading control. Protein signals were detected using an enhanced chemiluminescence (ECL) solution (Thermo Fisher Scientific (Waltham, MA, USA); 32106). Chemiluminescence was then measured in the LAS-3000 mini biomolecular imager.
8
Flow Cytometry Antibody Staining Protocol
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Antibodies (Abs) for flow cytometry were purchased from eBioscience (anti-B220, −CD3, −CD4, −CD11b, −F480, and − CD8), BD Biosciences (anti-CCR5, −Annexin V, and -BrdU), and Cell Signaling Technology (anti-Caspase-3 Asp175). Staining processes were performed according to the manufacturer’s instruction. Briefly, for cell surface straining, cells were blocked with Fc γ MAb (0.5 μg/ml) for 30 min at 4°C. After being washed with PBS, cells were stained with antibodies against B220, CD3, CD4, CD11b, F480, CD8 or CCR5 for 30 min on ice with gentle shaking. For cell apoptosis analysis, fresh cells were stained with anti-Annexin V antibody and propidium iodide (PI), while pre-fixed and permeated cells were stained using anti-caspase-3 antibody. For BrdU incorporation assay, the RABV-infected mice (n ≥3) were i.p. injected with BrdU (10 mg/ml, 100 μl) for 24 hours (hrs); cells from brains were then collected. After being stained with cell surface markers for 30 min on ice, the cells were stained with anti-BrdU antibody according to the manufacturer’s instruction. Samples were processed using FACSCalibur or Accuri C6 (BD Biosciences, San Jose, CA, USA), and data were analyzed with the FlowJo software (Tree Star, Ashland, OR, USA).
9
EV Characterization by Immunoblotting
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Transfected cells were washed in PBS and lysed directly into 4 × Laemmli buffer (#1610747, Biorad, Watford, UK). Isolated EVs were lysed directly in 4 × Laemmli buffer (#1610747, Biorad). Immunoblots were performed as previously described [46 (link)]. All antibodies were purchased from Cell Signalling (NEB, Hitchin, UK) and were used at 1:1000 dilution: Dicer (D38E7, #5362 S), Beta-Tubulin (9F3, #2128 S), Annexin V (#8555 S), Alix (3A9, #2171 S), CD54/I-CAM (#4915 S), EpCAM (D1B3, #2626 S).
10
Western Blot Analysis of Exosomal and Cellular Proteins
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Exosomes (10 μg) or proteins (30–50 μg) extracted from cells were diluted 1:4 (v/v) in a loading buffer (Beyotime, China), heated at 95°C for 10 min, loaded onto 10% gels (1.5 × 10 wells; Genscript, China), electrophoresed at 180 V for 50 min, and transferred to membranes by using the iBlot (Invitrogen) 8‐min program. For all subsequent antibody‐incubation and washing steps, a rocking platform was used. After blocking with a blocking buffer (Odyssey, USA) for 60 min, the membranes were incubated with primary antibodies at 4°C overnight, washed in TBST (3 × 5 min), incubated with secondary antibodies at room temperature for 60 min, washed with TBST, and then exposed to a detection reagent (Chemiluminescent HRP Substrate, Millipore, USA) for visualization. The primary antibodies were as follows: CD9 (Cell Signaling Technology, USA, 1:1000), Alix (Cell Signaling Technology, 1:1000), Annexin V (Cell Signaling Technology, 1:1000), HSP70 (Cell Signaling Technology, 1:1000), GM130 (Cell Signaling Technology, 1:1000), COL1A1 (Cell Signaling Technology, 1:1000), GAPDH (Cell Signaling Technology, 1:1000), β‐Actin (Cell Signaling Technology, 1:1000), and RANKL (Proteintech, China, 1:3000).
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