Alexa fluor 488 labeled annexin 5

Manufactured by Thermo Fisher Scientific

Alexa Fluor 488-labeled annexin V is a fluorescent dye-labeled protein used for detecting and quantifying apoptosis (programmed cell death) in cells. Annexin V binds to phosphatidylserine, which is exposed on the cell surface during apoptosis. The Alexa Fluor 488 dye provides a green fluorescent signal that can be detected and measured using flow cytometry or fluorescence microscopy.

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4 protocols using alexa fluor 488 labeled annexin 5

Annexin V and Propidium Iodide Assay

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Following incubation in the presence or absence of CL-ND, TAZ knockdown and control HL60 cells were incubated with a) Alexa Fluor 488-labeled annexin V (Invitrogen) or b) propidium iodide (PI), as reported by Riccardi and Nicoletti [26 (link)]. In both assays, the cells were subject to flow cytometry analysis on a BD LSRFortessa and the data processed using FlowJo software.

Ikon N., Su B., Hsu F.F., Forteand T.M, & Ryan R.O. (2015). Exogenous cardiolipin localizes to mitochondria and prevents TAZ knockdown-induced apoptosis in myeloid progenitor cells. Biochemical and biophysical research communications, 464(2), 580-585.

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Synthesis and Purification of Modified Nucleosides

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Phosphate-buffered saline (PBS), antibiotic and antifungal agents, amphotericin, propidium iodide, PrestoBlue, DAPI, Alexa Fluor 488, and apoptosis assay kit containing Alexa Fluor 488-labeled Annexin V were from Invitrogen. 3-Eth-5-NIdR and 3-Eth-5-NITP were synthesized and purified as previously described [13 (link), 15 (link)]. DNA including that containing an abasic site were obtained from Operon and purified as described [13 (link), 15 (link)]. Recombinant human polymerase delta (pol δ) and human polymerase eta (pol η) were purified as previously described [35 (link), 36 (link)]. Each polymerase was judged to be > 97% pure as assessed by sodium dodecylsulfate-polyacrylamide denaturing gel electrophoresis.

Choi J.S., Kim S., Motea E, & Berdis A. (2017). Inhibiting translesion DNA synthesis as an approach to combat drug resistance to DNA damaging agents. Oncotarget, 8(25), 40804-40816.

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Lipid Membrane Composition Analysis

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POPC, egg SM (eSM), Cholesterol (CHOL), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rho-DOPE) were from Avanti Polar Lipids (Alabaster, AL). 1,6-diphenyl-1,3,5-hexatriene (DPH) was from Sigma-Aldrich (St. Louis, MO). Octydecylrhodamine B (ODRB), 3,3′-dilinoleyloxacarbocyanine perchlorate (FAST DiO), dithiothreitol (DTT), and 4 (w/v) % PFA were from Invitrogen (Carlsbad, CA). All lipids and lipid probes were stored at −20°C. NEM was from Sigma-Aldrich dissolved in methanol and stored as aliquots at −20°C. Proteinase K (ProK) was from Thermo Fisher (Bohemia, NY). Phenylmethylsulfonyl fluoride (PMSF) was from Gold Biotechnology (St. Louis, MO). Octyl glucoside (OG) was from Anatrace (Maumee, OH) and dissolved in water. 10× phosphate-buffered saline (PBS) was from Bio-RAD (Hercules, CA). Dulbecco’s PBS (DPBS) (200 mg/l KCl, 200 mg/l KH₂PO₄, 8 g/l NaCl, and 2.16 g/l Na₂HPO₄) was from Thermo Fisher Scientific (Waltham, MA). Alexa Fluor 488 labeled Annexin V was from Life Technologies (Carlsbad, CA) and 5× annexin binding buffer from Thermo Fisher Scientific. All other chemicals were reagent grade.

Kakuda S., Suresh P., Li G, & London E. (2021). Loss of plasma membrane lipid asymmetry can induce ordered domain (raft) formation. Journal of Lipid Research, 63(1), 100155.

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Quantitative Apoptosis Assessment via Annexin V

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For quantitative assessment of apoptosis, the cells were incubated in Annexin V binding buffer containing 20-fold diluted Alexa Fluor 488-labeled Annexin V (Life Technologies/Thermo Fisher Scientific) and 5 μM Hoechst33342 (Life Technologies/Thermo Fisher Scientific) for 3 min. At least 3 separate fields of view were acquired for each condition by confocal microscopy using an UPLAPO 40× (0.85) dry objective. Annexin V positive cells and total cell number were determined on the basis of green and blue fluorescence, respectively. The results were expressed as percentage of total cell number. For co-staining of apoptotic cells and ABCG4, the cells were first subjected to Alexa Fluor 647- conjugated Annexin V (Life Technologies/Thermo Fisher Scientific) (20-fold dilution, 3 min), then fixed and immunostained as described above. To detect caspase-3 activity and Annexin V positivity in same experiment, the cells were incubated with 10 μM PhiPhiLuxG2D2 (Calbiochem) and 10% FCS for 20 min, washed twice, then subjected to Alexa Fluor 488-labeled Annexin V in Annexin V binding buffer for 3 min. Results are expressed as mean ± S.E.M. For statistical analysis, Student's t-test was used to evaluate significant differences. (p < 0.01) in comparison with controls.

Hegyi Z, & Homolya L. (2016). Functional Cooperativity between ABCG4 and ABCG1 Isoforms. PLoS ONE, 11(5), e0156516.

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