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Alexa488 beads

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Alexa488 beads are fluorescent particles used as calibration standards in flow cytometry and microscopy applications. These beads emit green fluorescence when excited by a 488 nm light source. They are designed to provide a consistent, quantifiable fluorescent signal for instrument setup, performance verification, and data analysis.

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Lab products found in correlation

Lsr 2 flow cytometer, by BD (4 mentions) Cd14 apc cy7, by BD (2 mentions) Cd66b pacific blue, by BioLegend (2 mentions) Cd3 alexa fluor 700, by BD (2 mentions) Human thp 1 cells, by American Type Culture Collection (1 mentions) Lsr 2, by BD (1 mentions)

6 protocols using alexa488 beads

1

Phagocytosis of ZEBOV Glycoprotein

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ZEBOV GPΔTM (IBT Biotherapeutics) was biotinylated and conjugated to streptavidin-coated Alexa488 beads (Life Technologies). Antibodies (5μg/ml) were incubated with beads for 2 hours at 37°C. Human monocytic cells (THP-1) were added at a concentration of 2.5 × 104 cells/well and incubated for approximately 18 hours at 37°C. Cells were fixed and analyzed by flow cytometry on a BD LSRII flow cytometer and a phagocytic score was determined as follows: (percentage of FITC+ cells) * (the geometric mean fluorescent intensity (MFI) of the FITC+ cells)/10,000.
Gunn B.M., Yu W.H., Karim M.M., Brannan J.M., Herbert A.S., Wec A.Z., Halfmann P.J., Fusco M.L., Schendel S.L., Gangavarapu K., Krause T., Qiu X., He S., Das J., Suscovich T.J., Lai J., Chandran K., Zeitlin L., Crowe JE J.r., Lauffenburger D., Kawaoka Y., Kobinger G.P., Andersen K.G., Dye J.M., Saphire E.O, & Alter G. (2018). A role for Fc function in therapeutic monoclonal antibody-mediated protection against Ebola virus.. Cell host & microbe, 24(2), 221-233.e5.
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2

Phagocytic Assay with RAVV GP

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RAVV GP was biotinylated and conjugated to streptavidin-coated Alexa488 beads (Life Technologies). Antibodies (range of concentration 5 μg/ml to 0.1 μg/ml) were incubated with beads for 2 hours at 37 °C. Human monocytic cells (THP-1) were added at a concentration of 2.5 × 104 cells/well and incubated for approximately 18 hours at 37 °C. Cells were fixed and analyzed by flow cytometry and a phagocytic score was determined using the percentage of FITC+ cells and the mean fluorescent intensity (MFI) of the FITC+ cells.
King L.B., Fusco M.L., Flyak A.I., Ilinykh P.A., Huang K., Gunn B., Kirchdoerfer R.N., Hastie K.M., Sangha A.K., Meiler J., Alter G., Bukreyev A., Crowe JE J.r, & Saphire E.O. (2018). The Marburgvirus-neutralizing human monoclonal antibody MR191 targets a conserved site to block virus receptor binding. Cell host & microbe, 23(1), 101-109.e4.
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3

Antibody-Mediated Phagocytosis Assay

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Recombinant BDBV GP was biotinylated and conjugated to streptavidin-coated Alexa488 beads (Life Technologies). BDBV-coated beads were incubated with antibodies at 5 μg/ml in culture medium for 2 hours at 37°C. Human white blood cells were isolated from peripheral blood by lysis of red blood cells using ammonium chloride potassium lysis buffer. Cells were washed with PBS, and 5.0 x 104 cells/well were added to bead-antibody immune complexes, and then incubated for 1 hour at 37°C. Cells were stained with the following antibodies to identify neutrophils: CD66b Pacific Blue (BioLegend clone G10F5), CD14 APC-Cy7 (BD Biosciences clone MφP9) and CD3 AlexaFluor700 (BD Biosciences clone UCHT1). Cells were fixed with 4% paraformaldehyde and were analyzed on a BD LSRII flow cytometer. A phagocytic score was determined as described above.
Ilinykh P.A., Santos R.I., Gunn B.M., Kuzmina N.A., Shen X., Huang K., Gilchuk P., Flyak A.I., Younan P., Alter G., Crowe JE J.r, & Bukreyev A. (2018). Asymmetric antiviral effects of ebolavirus antibodies targeting glycoprotein stem and glycan cap. PLoS Pathogens, 14(8), e1007204.
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4

Ebola Glycoprotein Phagocytosis Assay

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EBOV GP recombinant protein (IBT Bioservices) was biotinylated and
conjugated to streptavidin-coated Alexa488 beads (Life Technologies). EBOV
GP-coated beads were incubated with antibodies diluted in culture medium to
5μg/ml for 2 hours at 37°C. Human white blood cells were
isolated from peripheral blood by lysis of red blood cells using ammonium
chloride potassium (ACK) lysis buffer. Cells were washed with PBS, and 5.0
× 104 cells/well were added to bead-antibody immune
complexes, and incubated for 1 hour at 37°C. Cells were stained with
the following antibodies to identify neutrophils: CD66b Pacific Blue
(BioLegend clone G10F5), CD14 APC-Cy7 (BD Biosciences clone MφP9),
and CD3 AlexaFluor700 (BD Biosciences clone UCHT1). Cells were fixed with 4%
paraformaldehyde and were analyzed on a BD LSRII flow cytometer. Data was
analyzed using FlowJo software, and a phagocytic score was determined using
the following calculation: (% of AlexaFluor488+cells)*(AlexaFluor488 geometric mean fluorescent intensity (MFI) of
AlexaFluor488+ cells)/10,000.
Bornholdt Z.A., Herbert A.S., Mire C.E., He S., Cross R.W., Wec A.Z., Abelson D.M., Geisbert J.B., James R.M., Rahim M.N., Zhu W., Borisevich V., Banadyga L., Gunn B.M., Agans K.N., Wirchnianski A.S., Goodwin E., Tierney K., Shestowsky W.S., Bohorov O., Bohorova N., Velasco J., Ailor E., Kim D., Pauly M.H., Whaley K.J., Alter G., Walker L.M., Chandran K., Zeitlin L., Qiu X., Geisbert T.W, & Dye J.M. (2019). A two-antibody pan-ebolavirus cocktail confers broad therapeutic protection in ferrets and nonhuman primates. Cell host & microbe, 25(1), 49-58.e5.
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5

Bead-based Phagocytosis Assay for BDBV

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Recombinant BDBV GP was biotinylated and conjugated to streptavidin-coated Alexa488 beads (Life Technologies). BDBV-coated beads were incubated with antibodies at 5 μg/ml in culture medium for 2 hours at 37°C. Human THP-1 cells (ATCC) were added at a concentration of 2.5 x 104 cells/well and incubated for 18 hours at 37°C in 96-well plates. Cells were fixed with 4% paraformaldehyde and analyzed by flow cytometry on a BD LSRII using Diva software and FlowJo analysis software. The phagocytic score was determined using the following calculation: (% of AlexaFluor488+ cells)*(AlexaFluor488 geometric MFI of AlexaFluor488+ cells)/10,000.
Ilinykh P.A., Santos R.I., Gunn B.M., Kuzmina N.A., Shen X., Huang K., Gilchuk P., Flyak A.I., Younan P., Alter G., Crowe JE J.r, & Bukreyev A. (2018). Asymmetric antiviral effects of ebolavirus antibodies targeting glycoprotein stem and glycan cap. PLoS Pathogens, 14(8), e1007204.
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6

Antibody-Dependent Cellular Phagocytosis Assay

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ZEBOV GPΔTM (IBT Biotherapeutics) was biotinylated and conjugated to streptavidin-coated Alexa 488 beads (Life Technologies). The beads were incubated with 5 μg/ml for 2 (huADCP) or 1 (mADCP) hr at 37 °C before human (THP-1, 2.5 × 104 cells/well) or mouse (RAW264.7, 1.0 × 105 cells/well) monocytic cells were added and incubated for 18 (huADCP) or 1 (mADCP) hr at 37 °C. Following fixation, the cells were analyzed by flow cytometry using a BD LSR2 flow cytometer. Data were analyzed using FlowJo software. A phagocytic score describing relative phagocytic activity was calculated as: Phagocytic score = (%FITC+ cells x MFI of FITC+ cells)/10,000 where MFI is the mean fluorescence intensity of all FITC+ cells. Each phagocytic score was also compared to that for a non-specific/irrelevant antibody and a control reaction lacking antibody that were both performed in the same experiment to account for cell/donor variability.
Saphire E.O., Schendel S.L., Fusco M.L., Gangavarapu K., Gunn B.M., Wec A.Z., Halfmann P.J., Brannan J.M., Herbert A.S., Qiu X., Wagh K., He S., Giorgi E.E., Theiler J., Pommert K.B., Krause T.B., Turner H.L., Murin C.D., Pallesen J., Davidson E., Ahmed R., Aman M.J., Bukreyev A., Burton D.R., Crowe JE J.r., Davis C.W., Georgiou G., Krammer F., Kyratsous C.A., Lai J.R., Nykiforuk C., Pauly M.H., Rijal P., Takada A., Townsend A.R., Volchkov V., Walker L.M., Wang C.I., Zeitlin L., Doranz B.J., Ward A.B., Korber B., Kobinger G.P., Andersen K.G., Kawaoka Y., Alter G., Chandran K, & Dye J.M. (2018). Systematic analysis of monoclonal antibodies against Ebola virus GP defines features that contribute to protection. Cell, 174(4), 938-952.e13.
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