To obtain streptavidin-PE-conjugated tetramer, biotinylated Dd/NP166–174 monomeric protein was purified and tetramerized at an 8:1 molar ratio with streptavidin-PE (Invitrogen, San Diego, CA, USA) according to the manufacturer's protocol. At day 10 post challenge, each group of mice was sacrificed and tracheotomy was performed. The lungs were perfused with 5 mL of PBS with 10 U/mL heparin (Sigma) using a syringe with a 25-gauge needle. The lung tissues were homogenized with 3 mL of Iscove's Modified Dulbecco's Medium (IMDM) through 70-µm cell strainers to obtain single-cell suspensions. Centrifugation was conducted afterward, lymphocytes were resuspended in fresh IMDM and washed with fluorescence-activated cell sorting (FACS) buffer (0.5% FBS and 0.09% NaN_3 in PBS). After blocking with anti-mouse CD16/CD32 (BD Biosciences, Franklin Lakes, NJ, USA), cells were stained with anti-CD8a APC (clone 53-6.7, Biolegend, San Diego, CA, USA), anti-CD44 FITC (clone IM7, Biolegend), and Dd/NP(I) tetramer-PE or Dd/NP(V) tetramer-PE. After staining, the cells were washed and fixed with FACS lysing solution for 15 minutes at RT in the dark. Cells were washed with FACS buffer twice and stored at 4℃ in the dark until FACS analysis.
Streptavidin pe
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Streptavidin-PE is a conjugate of the protein streptavidin and the fluorescent dye phycoerythrin (PE). Streptavidin has a high affinity for the small molecule biotin, which allows it to be used in a variety of bioanalytical techniques that involve biotinylated molecules. The PE moiety provides fluorescent labeling capabilities for the detection and quantification of biotinylated targets.
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Lab products found in correlation
Streptavidin apc, by Thermo Fisher Scientific (15 mentions)
Facscanto 2, by BD (7 mentions)
Lsrfortessa, by BD (6 mentions)
Facsaria 2, by BD (6 mentions)
Facscalibur, by BD (4 mentions)
S1300exi flow cytometer, by Stratedigm (3 mentions)
Lsr 2, by BD (3 mentions)
7 aad, by BD (3 mentions)
Annexin 5 pe, by BD (3 mentions)
Streptavidin bv421, by BioLegend (3 mentions)
Golgiplug, by BD (3 mentions)
Mouse ccl24, by Thermo Fisher Scientific (2 mentions)
Biotinylated mccl11, by Merck Group (2 mentions)
Anti cd4 buv395, by BD (2 mentions)
Dasatinib, by Selleck Chemicals (2 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (2 mentions)
Bio plex 200 system, by Bio-Rad (2 mentions)
Gr1 biotin, by BD (2 mentions)
Apc labeled anti brdu mab 3d4, by BD (2 mentions)
Streptavidin percp and apc, by BD (2 mentions)
135 protocols using streptavidin pe
1
Quantifying Antigen-Specific CD8+ T Cells
2
Tetramerization of sTCR with Streptavidin-PE
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For tetramerization with streptavidin-PE (Invitrogen), the ratio of sTCR:streptavidin-PE was 4:1. 4.07-µg sTCR were added to 4-µl streptavidin-PE and incubated for 45 min on ice. Afterwards, 1.325 µl of the tetramer suspension was used to stain up to 1 × 106 cells per sample in PBS + 1% FCS. After 20 min on ice, the cells were washed once with PBS + 1% FCS and the fluorescence detected by flow cytometry.
3
Biotinylated MHC Tetramer Crosslinking
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Streptavidin-PE (Life Technologies) was added three times at 30 min intervals to biotinylated H-2Kb or H-2Db monomers to a final molar ratio of 4:1 for monomer:Streptavidin-PE. Tetramers were stored at 4°C and used within 1 month. Tetramers were diluted to 50 µg/ml in DMEM without phenol red (Life Technologies) and supplemented with 10% fetal bovine serum (FBS) (Hyclone, South Logan, UT, USA), 1% Penicillin/Streptomycin (Gibco, Grand Island, NY, USA), 1% GlutaMAX (Gibco), and 0.1% NaN3 (Sigma, St. Louis, MO, USA). The solution was deposited in a 96-well plate (V-bottom, 110 µl per well), and index peptide was added (2 µl of 10 mM peptide per well). The plate was placed on ice and irradiated for 30 min in a CL-100 UV Crosslinker (UVP, Upland, CA, USA) equipped with 365 nm UV-lamps at a ≈10–20 cm distance. After 1 h of incubation at 37°C, the plate was centrifuged (3,000 × g) for 20 min, and 50 µl of the supernatant was transferred into new 96-well plate and used for cell surface staining (see Tetramer Staining and Flow Cytometry ).
4
Quantifying Eosinophil CCL11 Binding
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A million bmEos were pre-treated with mouse CCL24 (Peprotech) at 200 ng/mL for 15 minutes on ice. Then, 20 ng/mL of biotinylated-mCCL11 (E8403, Sigma) was added for 30 minutes. Cells were then incubated in the presence of streptavidin-PE (Invitrogen) at 1:1000 and assessed by FACS.
5
ACE2-RBD Binding Assay by Flow Cytometry
6
Generating H-2Ks Tetramers for MHC Binding
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H-2Ks tetramers were generated as previously described [17 (link)]. Briefly, H-2Ks and the human β2-microglobulin gene were subcloned into the pET28 bacterial expression vector. BL21/DE3 competent cells were transformed and protein expression was induced with IPTG for 4–5 h. Inclusion bodies were purified and refolded in the presence of peptides. The soluble monomeric form of the peptide-MHC complex was biotinylated with BirA at room temperature and tetramerized with streptavidin-PE (Invitrogen, Carlsbad, CA, USA).
7
Biotin-labeled SARS-CoV-2 and SARS-CoV RBD analysis
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Biotinylated SARS‐CoV‐2 and SARS‐CoV RBDs were produced by co‐transfection of Avi/His‐tagged RBD expression plasmids with an expression plasmid encoding BirA enzyme. RBD proteins were purified from transiently‐transfected Freestyle 293F cell (Gibco) supernatants by nickel affinity and size‐exclusion chromatography. To prepare fluorochrome‐conjugated streptavidin‐tetramerized RBDs, biotinylated SARS‐CoV‐2, and SARS‐CoV RBDs were incubated with streptavidin‐A488 (Invitrogen) and streptavidin‐PE (Invitrogen), respectively, overnight at 4 °C at a 1:4 molar ratios of RBD to streptavidin subunit. For analysis of RBD‐specific B cells, the 1 × 106 cells in the draining lymph nodes were stained at 4 °C for 1 h with 6 µg SARS‐RBD‐A488, 4 µg SARS‐2‐RBD‐PE, and the following antibodies the viability maker (FVS 440), anti‐CD45‐APC‐Cy7, anti‐CD19‐PE‐Cy7, anti‐CD38‐BV605, anti‐GL7‐BV421 at 4 °C for 15 min to acquire the stained cells.
8
Staining CD8+ T Cells with pHLA Tetramers
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p‐HLA tetramers were prepared by conjugating purified biotinylated p‐HLA monomers to streptavidin at a 8:1 monomer to streptavidin molar ratio. Streptavidin‐PE (Invitrogen, Waltham, USA) was added slowly onto the monomer at 1/10 of the volume required and incubated for 10 min at room temperature, 10 times. CD8+ T cell lines were tetramer stained for 1 h at room temperature. Cells were washed and surface stained with anti‐CD3‐BV480 (BD Biosciences), anti‐CD8‐PerCP‐Cy5.5 (BD Biosciences), anti‐CD4‐BUV395 (BD Biosciences) and live/dead fixable near‐IR dead cell stain (Life Technologies). Cells were fixed with 1% paraformaldehyde and acquired on the BD LSR Fortessa and were analysed using Flowjo v10 (BD Biosciences). The gating strategy is shown in Supplementary figure 1b .
9
MHC Tetramer Staining Protocol
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MHC tetramer staining was performed as previously described(47 (link)). Briefly, MHC tetramer reagents were non-covalently linked to streptavidin-PE (Invitrogen, cat#: S866) and/or streptavidin-APC (Invitrogen, cat#: S868) 1:1 mol:mol. Single-cell suspension of total PBMC/splenocytes or isolated CD8+ T cells were suspended in PBS (FCS 2% w/v, EDTA 2mM) supplemented with dasatinib (SelleckChem, cat#: S1021) 50nM, then incubated 30min, 20°C. MHC-tetramer (100nM), and anti-mouse CD8a (2.5× 10−4)g/L, Clone: CT-CD8a) was added then incubated, 60min, 4°C. The cells were then washed with PBS (FCS 2%, EDTA 2mM) twice, then suspended in PBS (Paraformaldehyde, 1% w/v) and stored at 4°C until use.
10
Tetramerization of CD1 Lipid Antigens
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Human CD1a, CD1b, CD1c, and CD1d monomers were obtained from the National Institute of Health (NIH) tetramer facility. For CD1a-endo, CD1b-endo, CD1c-endo, and CD1d-endo tetramers the monomers were used at 0.2 mg/ml in TBS and tetramerized. Synthetic dideoxymycobactin (3.0 μg) was dried in a 10 mm wide glass tube and dissolved in 6 ml dimethylsulfoxide (DMSO), followed by addition of 90 ml 2% CHAPS in Tris buffered saline and sonication for 1 h at 37 °C. After a short spin, 90 ml was transferred to a plastic tube, followed by addition of 20 μg CD1a monomer and incubation for 2 h at 37 °C. Monomers were tetramerized using streptavidin-APC (Molecular Probes) or streptavidin-PE (Invitrogen).
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