Using the assay developed by Ackerman et al. (2017) (link) (109), 5 μg of V1V2(ZM53)-2F5K, V1V2(ZM109)-1FD6, or V1V2(ZM109)-TTB were biotinylated using EZ-Link Sulfo-NHS-LC-LC-Biotin (Thermo Scientific) and then conjugated to fluorescent neutravidin beads (Thermo) according to manufacturer’s instructions. Conjugated beads were washed and resuspended in 0.1% BSA-PBS to a working dilution of 1:100. Nine hundred thousand (9 × 105) beads were aliquoted per well in round bottom 96-well plates. Four-fold dilutions of plasma obtained at week 22 or at late time points after immunization of the NHPs were titrated, added to beads, incubated for 2 hr at 37°C, and washed. Twenty-five thousand (2.5 × 104) THP-1 cells (ATCC, Cat. No. TIB-202, RRID:CVCL_0006) were added to each well and were incubated overnight. Phagocytosis was measured by flow cytometry. Antibody-dependent cellular phagocytosis (ADCP) scores were calculated as (% bead-positive cells × mean fluorescence intensity [MFI] of bead-positive cells)/106. Area under the curve (AUC) was calculated from the titration curves using GraphPad Prism.
Fluorescent neutravidin beads
Manufactured by Thermo Fisher Scientific
Fluorescent neutravidin beads are small, spherical particles coated with the protein neutravidin, which has a high affinity for biotin. These beads are labeled with fluorescent dyes, allowing them to be easily detected and tracked in various biological applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa, by BD (5 mentions)
Sulfo nhs lc biotin, by Thermo Fisher Scientific (3 mentions)
Ique screener plus, by Intellicyt (3 mentions)
Thp 1 cell, by American Type Culture Collection (3 mentions)
Ez link nhs lc lc biotin, by Thermo Fisher Scientific (2 mentions)
Ez link sulfo nhs lc lc biotin, by Thermo Fisher Scientific (1 mentions)
Nhs sulfo lc lc kit, by Thermo Fisher Scientific (1 mentions)
Ique analyzer, by Intellicyt (1 mentions)
Zeba spin desalting column, by Thermo Fisher Scientific (1 mentions)
Thp 1 effector cells, by American Type Culture Collection (1 mentions)
Flow cytometer s100exi, by Stratedigm (1 mentions)
Gvb buffer, by Boston BioProducts (1 mentions)
Fluorescein isothiocyanate (fitc), by MP Biomedicals (1 mentions)
Lsrii instrument, by BD (1 mentions)
Negative selection, by STEMCELL (1 mentions)
Ez link sulfo nhs lc lc biotin kit, by Thermo Fisher Scientific (1 mentions)
S1000exi flow cytometer, by Stratedigm (1 mentions)
Paraformaldehyde solution, by Santa Cruz Biotechnology (1 mentions)
Lsr 2, by BD (1 mentions)
Facsaria 2, by BD (1 mentions)
28 protocols using fluorescent neutravidin beads
1
Antibody-Dependent Cellular Phagocytosis Assay
2
Quantifying Neutrophil Phagocytosis Capacity
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Phagocytosis score of primary human neutrophils was determined as described before.30 (link) Antigens were biotinylated with NHS-Sulfo-LC-LC kit according to the manufacturer’s instruction (Thermo Fisher). Excessive biotin was removed by size exclusion chromatography using Zeba-Spin desalting columns (7 kDa cutoff, Thermo Fisher). Biotinylated antigens were coupled to fluorescent neutravidin beads (Thermo Fisher) and incubated with 1:10 diluted plasma. Primary cells were derived from Ammonium-Chloride-Potassium (ACK) buffer lysed whole blood from healthy donors and incubated with immune complexes for one hour at 37°C. For the Fc-receptor blocking experiments, isolated neutrophils were pre-incubated with 5 μg/ml of FcγR2a (CD32A, clone IV.3, Bio X Cell Cat# BE0224, RRID:AB_2687707 ) and FcγR3 (CD16, clone: LNK16, Bio-Rad, RRID:AB_324304 ) five minutes prior to addition of neutrophils to the immune complexes. Neutrophils were stained for surface CD66b (BioLegend Cat# 305112, RRID:AB_2563294 ) expression, fixed with 4% para-formaldehyde, and analyzed an iQue analyzer (IntelliCyt) (Figure S8 ).
3
Phagocytosis Assay with Biotinylated Protein
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This assay was performed as described previously.63 (link) In short, Fluorescent NeutrAvidin beads (Thermo Scientific) were incubated overnight at 4°C with biotinylated GPC-I53-50A protein (10μg/5μL beads suspension). After incubation, the beads were washed twice using PBS 2% bovine serum albumin (BSA). 50 μL of the bead suspension were placed in a V-bottom 96-well plate and incubated with 10-fold serial dilutions of guinea pig serum at a start dilution of 1:1000 in PBS 2%BSA. After 2 h at 37°C, the plates were washed and 5×104 THP-1 effector cells (monocytes; ATCC) were added to each well. To promote beads to cell contact, plates were quickly spun down before incubation for 5 h at 37°C. After incubation, the cells were washed and resuspended in PBS 2% FCS. Cells were analyzed by flow cytometry and the phagocytic activity was determined by the area under curve of the MFI (beads positive cells x mean MFI FITC).
4
Neutrophil Phagocytosis Assay with IgA-Depleted Plasma
5
Antibody-Mediated Neutrophil Phagocytosis Assay
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Antibody-dependent neutrophil-mediated phagocytosis was assessed by the measurement of the uptake of antibody-opsonized, antigen-coated fluorescent beads by primary neutrophils26 (link). Biotinylated A244 gp120 was used to saturate the binding sites on 1 μm fluorescent neutravidin beads (ThermoFisher). Excess antigen was removed by washing the beads, which were then incubated with animal Ab samples for 20 min at 37 °C. Leukocytes were isolated from blood collected from HIV-seronegative donors by ACK lysis of red blood cells. Following opsonization, the freshly isolated leukocytes were added, and the cells were incubated for 1 h at 37 °C to allow phagocytosis. The cells were then stained for CD66b (BioLegend #305112; 1 μl per test) to identify neutrophils and fixed, and the extent of neutrophil phagocytosis was measured via flow cytometry (gating on CD66b-positive cell) on a Stratedigm S100EXi flow cytometer equipped with high-throughput sampler. The data are reported as a phagocytic score, which takes into account the proportion of effector cells that phagocytosed and the degree of phagocytosis (integrated MFI: frequency × MFI).
6
Complement Deposition Assay Protocol
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Complement deposition was performed as described before (45 (link)). In brief, biotinylated antigens were coupled to fluorescent neutravidin beads (Thermo Fisher) and incubated with 10 μl of 1:10 diluted plasma. Beads were incubated with guinea pig complement in GVB++ buffer (Boston BioProducts, MA, USA) for 20 min at 37°C. Deposited C3 on beads was stained with anti-guinea pig C3 antibody labeled with fluorescein isothiocyanate (FITC) (MP Biomedicals, CA, USA) and analyzed by flow cytometry on a LSRII instrument (BD Biosciences, CA, USA).
7
THP-1 Phagocytosis of Biotinylated Antigens
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THP-1 phagocytosis assay was performed as described before (47 (link)). In brief, biotinylated antigens were coupled to fluorescent neutravidin beads (Thermo Fisher) and incubated with 50 μl of 1:200 diluted plasma. THP-1 monocytes were added to the beads, incubated for 16 h at 37°C, fixed with 4% paraformaldehyde, and analyzed by flow cytometry on a LSR Fortessa (BD).
8
Neutrophil Phagocytosis Assay with Immune Complexes
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Phagocytosis score of primary human neutrophils was determined as described before (46 (link)). Biotinylated antigens were coupled to fluorescent neutravidin beads (Thermo Fisher) and incubated with 50 μl of 1:200 diluted plasma. Primary cells were derived from ammonium-chloride-potassium (ACK) buffer-lysed whole blood from healthy donors and incubated with immune complexes for 1 h at 37°C. Neutrophils were stained for surface CD66b (Biolegend, CA, USA; clone G10F5) expression, fixed with 4% paraformaldehyde and analyzed by flow cytometry on a LSR Fortessa (BD).
9
Multiplex Functional Immunoassay Protocol
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For the functional assays, antigens were biotinylated and coupled to fluorescent neutravidin beads (Thermo Fisher). Beads were incubated with plasma samples to form immune complexes in 96-well plates for 2 h at 37 °C. Immune complexes were washed and incubated with guinea pig complement factor for 20 min at 37 °C (for antibody-dependent complement deposition (ADCD)), primary neutrophils from ACK lysed blood for 1 h at 37 °C (for antibody-dependent neutrophil phagocytosis (ADNP)) or with THP-1 monocytes for 16 h at 37 °C (for ADNP). ADCD was detected on beads using a polyclonal anti-guinea pig C3-FITC antibody (ADCD) and neutrophils stained for CD66 expression using a respective antibody (Biolegend) after the respective incubation. All samples were fixed with 4% para-formaldehyde prior to analysis on an Ique analyzer.
10
ADCP and ADNP Antibody Assays
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ADCP and ADNP experiments were performed as previously described 18 (link),19 (link). Briefly, antigen proteins of the target were biotinylated using the EZ-link™Sulfo-NHS-LC-LC-Biotin kit (Thermo Fisher), then coupled to the fluorescent neutravidin beads (Thermo Fisher, F8776). The bead-antigen conjugates were incubated with diluted serum and BAL samples (serum: 1:100, BAL: 1:10) for 2 h at 37°C. The unbound antibody was removed by washing buffer. The immune complexes were then incubated overnight with cultured THP-1 cells (ADCP), or for 1 h with primary neutrophils isolated from human whole blood (ADNP) using negative selection (Stemcell). Treated THP-1 cells were subsequently washed and fixed in 4% paraformaldehyde (PFA), while the treated neutrophils were washed, stained for CD66b+ marker (Biolegend), and fixed in 4% (PFA) prior to flow cytometry analysis. A phagocytosis score for THP-1 or neutrophil was eventually determined as (% cells positive × Median Fluorescent Intensity of positive cells). Flow cytometry was performed with an iQue (IntelliCyt) instrument and population measurements were conducted using IntelliCyt ForeCyt (v8.1). The reagents and materials used are listed in the Key Resources Tables .
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