RBD protein expressed with AviTag was purchased from GenScript. Site-specific biotinylation of the AviTag was performed using BirA Biotin-Protein Ligase Reaction kit (Avidity). Next, unconjugated biotin was removed using Zeba spin desalting columns, 7K MWCO (ThermoFisher). The quantification of reacted biotin was performed using the Pierce Biotin Quantification Kit (ThermoFisher). Biotinylated RBD was incubated with either streptavidin-conjugated PE (Biolegend) or streptavidin-conjugated APC fluorophores (Biolegend) for 20 min on ice at a molar ratio of 4:1 of biotin to streptavidin. Streptavidin-conjugated FITC (BioLegend) was reacted with excess free biotin to form a non-RBD-specific streptavidin probe as a control. Tetramer formation was confirmed using SDS-PAGE gel. Cells were stained for flow cytometry with all three streptavidin probes at the same time as other fluorescent surface markers at a volumetric ratio of 1:100 for RBD-streptavidin-PE and 1:200 for RBD-streptavidin-APC and biotin-streptavidin-FITC.
Fitc conjugated streptavidin
Manufactured by BioLegend
Sourced in Morocco, United States
FITC-conjugated streptavidin is a fluorescent conjugate used to detect biotinylated molecules. Streptavidin, a bacterial protein, binds strongly to biotin. The FITC fluorescent dye is covalently attached to the streptavidin, allowing visualization of biotinylated targets.
Automatically generated - may contain errors
Lab products found in correlation
Pe conjugated streptavidin, by BioLegend (3 mentions)
Avitag, by GenScript (1 mentions)
Pierce biotin quantification kit, by Thermo Fisher Scientific (1 mentions)
Streptavidin conjugated apc fluorophores, by BioLegend (1 mentions)
Avitag, by Avidity (1 mentions)
Bira biotin protein ligase reaction kit, by Avidity (1 mentions)
Ez link nhs biotin, by Thermo Fisher Scientific (1 mentions)
Quanttag biotin quantification kit, by Vector Laboratories (1 mentions)
Eflour 670, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Facscalibur machine, by BD (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Eclipse te2000 u microscope, by Nikon (1 mentions)
Dapi fluoromount g, by Southern Biotech (1 mentions)
Jc 1 dye, by Thermo Fisher Scientific (1 mentions)
Annexin 5, by BioLegend (1 mentions)
Biotinylated annexin 5, by BioLegend (1 mentions)
Cytoflex s, by Beckman Coulter (1 mentions)
Cy3 conjugated streptavidin, by Jackson ImmunoResearch (1 mentions)
8 protocols using fitc conjugated streptavidin
1
Biotinylated RBD Tetramer Assay
2
Biotinylated Uricase Multimer Assay
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Uricase was biotinylated using EZ-link NHS-biotin (Thermo Scientific). Unreacted NHS-biotin was removed using Zeba spin desalting columns, 7kDa MWCO (Thermo Scientific). The extent of biotinylation was measured using the QuantTag Biotin Quantification kit (Vector Laboratories) to ensure 1:1 M ratio of uricase and biotin. Biotinylated uricase was reacted for 20 min on ice with 4:1 M ratio of biotin to streptavidin-conjugated PE or streptavidin-conjugated APC (Biolegend). Streptavidin-conjugated FITC (BioLegend) was reacted with excess free biotin to form a non-antigen-specific streptavidin probe as a control. Multimer formation was confirmed using SDS-PAGE gel. Cells were stained for flow cytometry with all three streptavidin probes at the same time as other fluorescent surface markers at a volumetric ratio of 1:200. The antigen-specific staining of B cells with multimer was verified by staining with and without 5M uricase on splenocytes from vaccinated and antigen-naive mice.
3
Proliferation and Lectin Staining Protocol
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For proliferation studies, cells were labelled with 10 μM proliferation dye eFlour 670 (eBiosciences, #65-0840-90) for 10 min on ice. Labelling was stopped by adding 4-5 volumes of complete culture medium for 5 min on ice. Cells were then subjected to flow cytometry analysis (day0) or maintained in culture for additional 5 days prior to analysis by Flow Cytometry. For lectin staining, Peanut Agglutinin (PNA, #B-1075), Sambucus Nigra Lectin (SNA, #B-1305), Aleuria Aurantia Lectin (AAL, #B-1395) and Maackia Amurensis Lectin II (MAA, # B-1265-1), all from vector laboratories, were used. Cells were blocked in carbon-free blocking buffer (vector laboratories, #SP-5040) for 20 min on ice, and then incubated with biotinylated lectins diluted in PBS containing 5% carbon-free blocking buffer. Lectins were then detected with FITC-conjugated streptavidin (Biolegend, #405201), Alexa Flour 647-conjugated streptavidin (Biolegend, #2068269) or PE-conjugated Streptavidin (Biolegend, #410504) in PBS for 20 min at 4°C. To differentiate between live and dead cells, all samples were stained with DAPI (Sigma, #32670, dilution 1:1000) prior to data acquisition. Data were acquired using an LSR II flow cytometer (BD) and analysed using FlowJo version 10.8.0.
4
Characterizing IL-22 Surface Expression on Lactobacillus
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Cell wall display of mouse IL-22 on the surface of Lactobacillus was confirmed by flow cytometric analysis. Fifty μl of Lactobacillus (Lp pAFβ900-IL22) cultures grown in MRS to an OD600 of 1.0 were pelleted by centrifugation (3000×g for 10 min) and washed twice in PBS. Bacteria were incubated with biotinylated goat anti-mouse IL-22 antibody (1 μg/ml) for 30 min on ice. After two PBS washes, the cells were incubated with FITC conjugated streptavidin (BioLegend, San Diego, CA, 1:500 dilution) on ice for 30 min in the dark. The antibody and conjugated streptavidin were diluted in PBS containing 1% BSA. After washing three times with PBS, samples were resuspended and fixed in 400 μl 1% paraformaldehyde and analyzed using a FACS Calibur machine (Becton–Dickinson, Franklin Lakes, NJ).
5
Time- and Dose-Dependent Biotin-E5 Binding Assay
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Leukemia cells of 5 × 105/well (HL-60, NB4, THP-1 and U937) were seeded in 24-well plates in 500 µL serum-free medium (opti-MEM; Life Technologies, Grand Island, NY). The stock solution of biotin-E5 was dissolved in the sterile water. In the time-effect assay, cells were incubated with biotin-E5 at 10 µM (37°C, 5% CO2) and collected at indicated time points (0–4 h). In the concentration-effect assay, cells were incubated with biotin-E5 at different concentrations (0–60 µM) for 2 h. Cells were then washed with BSA/PBS and incubated with FITC-conjugated Streptavidin (BioLegend, San Diego, CA) at 4°C. After washed with BSA/PBS, the cells were subjected to C6 Accuri® flow cytometer. The 1.5 × 104 cells were collected, acquired data were analyzed by CFlow Plus software.
6
Hyaluronan Detection in Activated MSCs
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We grew MSCs on 4 or 8-well Lab-Tek slide chambers (Nalge Nunc Int, Roskilde, Denmark) in 2% CCM at 5 × 103 cells/well and allowed the cells to adhere for at least 6 h. MSCs were then activated with Poly (I:C) or LPS for 24 h and cells washed with PBS and fixed in an acid-formalin-ethanol solution (3.7% formaldehyde-PBS, 70% ethanol and 5% glacial acetic acid, all v/v)51 (link). HA was detected using a biotin-conjugated HA binding protein (HABP) (4 μg/ml, Calbiochem, Billerica, MA) followed by FITC-conjugated streptavidin (1:1000, Biolegend, San Diego, CA). Coverslips were mounted with DAPI Fluoromount-G (Southern Biotech) to counterstain nuclei and slides were analyzed in a Nikon Eclipse TE 2000-U microscope (Nikon Instruments Inc., Melville, NY).
7
Apoptosis, Membrane Potential, and ROS Assay
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Apoptosis was measured by staining cells with Annexin V. Cells were washed with ice cold PBS and stained with biotinylated Annexin V (Biolegend) at suggested concentration for 15 min, washed, then incubated with FITC-conjugated streptavidin (Biolegend). Cells were washed with PBS extensively to remove non-specific binding. Membrane potential was measured by using JC-1 dye (ThermoFisher) as described in the product manual. Briefly, cells in DMEM medium were incubated with an equal volume of staining solution containing 5μg/ml JC-1 at 37° C for 20 min. Cells were washed for 3 times with PBS and resuspended in DMEM. ROS was measured by staining PBS-washed cells with 10 μM DCHF-DA at 37° C for 30 min in the dark. Cells were then washed with PBS and resuspended in DMEM. Flow cytometry analysis was performed on cytoFLEX S (BECKMAN). Data were analyzed with FlowJo V10.7.
8
Flow Cytometry Reagents for Immune Cell Analysis
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An fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody (mAb) to mouse CD8a (53-6.7); phycoerythrin (PE)-conjugated mAbs to mouse CD4 (RM4-5) and I-A (M5/114); and an allophycocyanin-or a biotin-conjugated mAb to mouse CD11c (HL3) were obtained from BD Biosciences (San Jose, CA, USA). PE-conjugated mAbs to F4/80 (BM8) and ESAM (1G8); a rat mAb to mouse CD16/32 (93); and biotin-conjugated mAbs to SIRPa (p84) and DCIR2 (33D1) were from eBioscience (San Diego, CA, USA). FITCconjugated streptavidin; PE-cyanine (Cy)7-conjugated streptavidin; a brilliant violet 421-conjugated mAb to mouse CD11b (M1/70); a brilliant violet 510-conjugated mAb to mouse CD8a (53-6.7); and an Alexa Fluor 488-conjugated mAb to mouse CD3e (145-2C11) were from BioLegend (San Diego, CA, USA). An Alexa Fluor 488-conjugated polyclonal Ab (pAb) to goat IgG was from Life Technologies. Cy3-conjugated streptavidin was from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Goat pAbs to mouse EBI2 (A20) were from Santa Cruz (Santa Cruz, CA, USA). Propidium iodide (PI) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Microbeads conjugated with mAbs to mouse CD11c (N418) were obtained from Miltenyi Biotech (Bergisch Gladbach, Germany).
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