Measurement of IgG subclass-specific pneumococcal antibodies was performed on sera collected from infants at 5 months of age using a previously described binding antibody multiplex assay [26 (link)]. Briefly, the 13 pneumococcal serotypes tested above were coupled to carboxylated fluorescent beads (Bio-Rad Laboratories, Inc., Hercules, CA, USA) following the DMTMM-coupling protocol [27 (link)]. The coupled beads were incubated with diluted plasma (at 1:100 dilution) for 30 minutes at 20°C. Antigen-specific subclass response was detected using biotin-conjugated mouse anti-human IgG1 (BD Pharmingen, San Diego, CA, USA; 4 μg/L), IgG2 (Southern Biotech, Birmingham, AL, USA; 5 μg/mL), IgG3 (Calbiochem, San Diego, CA, USA; 2 μg/mL), or IgG4 (BD Pharmingen; 2 μg/mL) and tertiary detection agent streptavidin-phycoerythrin (BD Biosciences, Franklin Lakes, New Jersey, USA) at 5 μg/mL. Beads were washed and read on a Bio-Plex 200 instrument (Bio-Rad Laboratories, Inc.). IgG levels were expressed as mean fluorescence intensity (MFI) and all MFI values were corrected by subtracting the intensity of a blank control. IgG levels below 100 MFI were considered to be below the lower limit of quantitation for the assay. Consistency between assays was measured by tracking the 50% effective concentration, MFI, and area under the curve of the positive control (007SP) by Levey-Jennings charts.
Streptavidin phycoerythrin
Manufactured by BD
Sourced in United States
Streptavidin-phycoerythrin is a fluorescent conjugate composed of the protein streptavidin and the fluorescent dye phycoerythrin. It is commonly used in various bioanalytical applications as a detection reagent due to the strong binding affinity between streptavidin and biotin.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (3 mentions)
Biotin conjugated mouse anti human igg1, by BD (2 mentions)
Carboxylated fluorescent beads, by Bio-Rad (1 mentions)
Bio plex 200 instrument, by Bio-Rad (1 mentions)
Ctla 4, by BioLegend (1 mentions)
Biotinylated anti egfr antibody, by R&D Systems (1 mentions)
Cd62l, by BD (1 mentions)
Cytoflex s, by Beckman Coulter (1 mentions)
Tim 3, by BioLegend (1 mentions)
Lsrfortessa, by BD (1 mentions)
Accuri c6, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Cflow plus software, by BD (1 mentions)
Sialidase, by Merck Group (1 mentions)
Rabbit anti mouse ng2, by Merck Group (1 mentions)
Rat anti mouse mhcii, by Thermo Fisher Scientific (1 mentions)
Rat anti mouse cd31, by Dianova (1 mentions)
Anti c5b 9 monoclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Alexa 647 conjugated secondary antibody, by Abcam (1 mentions)
Alexa 488 conjugated anti c3c antibody, by Abcam (1 mentions)
11 protocols using streptavidin phycoerythrin
1
Pneumococcal Antibody Subclass Profiling
2
Norovirus VLP Binding to Host Cells
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The binding of norovirus VLPs to cells in vitro was assessed using HT-29 (human colorectal adenocarcinoma cells), a cell line with well-characterized H antigen and A antigen expression. In addition, Chinese hamster ovary (CHO) cells transfected with rat α1,2-fucosyltransferase B cDNA, with or without cotransfection with the rat A enzyme cDNA or B enzyme cDNA, were utilized. The CHO cells were generated as part of a previous study (11 (link)). A total of 2.5 × 105 viable cells were incubated with 10 μg/ml VLPs in PBS-0.1% BSA for 1 h at 4°C. After 3 washes with the same buffer, a 30-min incubation was performed with anti-VLP antibody. After washes, a third incubation was performed with biotinylated anti-rat secondary antibody under the same conditions. The final incubation step used streptavidin-phycoerythrin (BD Pharmingen). After final washes in PBS alone, fluorescence analysis was performed on a FACSCalibur (Becton, Dickinson, Rungis, France) using the CELLQuest program. Blocking of VLP binding with synthetic oligosaccharides was achieved by preincubating 10 μg/ml VLPs with 400 μg/ml oligosaccharides for 1 h at 37°C. Fucosidase treatment of cells was achieved by incubation at 37°C with 10 μg fucosidase in 100 mmol/liter sodium phosphate buffer, pH 6.5, for 1 h.
3
Phenotypic Characterization of CAR-T Cells
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Untransduced- or CD19CAR-T cells were cultured with the γ-irradiated CD19-K562 cell line in a 1:1 ratio for seven days without IL-2 supplementation. The T-cells were stained with monoclonal antibodies conjugated with fluorophores: CD3, CD8, CD45RA, CD62L (BD Biosciences), PD-1, LAG-3, CTLA-4, and TIM-3 (BioLegend, San Diego, CA, USA). The tEGFR+ cells were stained with biotinylated anti-EGFR antibody (R&D Systems, Bio-Techne, MN, USA) and counterstained with streptavidin-phycoerythrin (BD Biosciences). All samples were analyzed by a CytoFlex S flow cytometer machine (Beckman Coulter Inc, CA, USA) and data were analyzed using FlowJo software (Tree Star).
4
Biotinylation and Tetramerization of CD1a
5
Tetramerized HLA-A*02:01/GLCTLVAML monomer for PBMC stimulation
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HLA-A*02:01/GLCTLVAML (A2-GLC) monomer (ImmunoID, University of Melbourne) was tetramerized with streptavidin-phycoerythrin (BD Biosciences, San Jose, CA, USA). For peptide pulsing, PBMCs were incubated with the GLC peptide (1–10 μm ; Genscript, Piscataway Township, NJ, USA) for 1–1.5 h at 37 °C, then washed twice before priming.30 (link), 31 (link) PBMCs were stimulated with GLC-pulsed autologous PBMCs for 10–13 days (37 °C, 5% CO2) at a 2:1 ratio in cRF10 media plus 10 U ml−1 recombinant interleukin-2.32 (link)
6
Immunophenotyping of Trypanosoma Parasites
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Tissue culture-derived trypomastigotes were collected and washed by centrifugation (2,000 g, 5 min) and resuspended in PBS. The parasites were fixed by adding the same volume of a 4% p-formaldehyde in PBS, which were maintained at 4°C until use. The fixed parasites were washed by centrifugation (2,000 g, 5 min) in a swing buckle rotor twice with PBS and resuspended to 2 × 10 7 /ml. One hundred microlitres of the culture supernatant of hybridoma mAb 3C9 (Andrews, Hong, Robbins, & Nussenzweig, 1987) diluted 5X, the ascitic fluid of mAb Mest-1 (Suzuki et al., 1997) diluted 100X in PBS, or Concanavalin-biotin (Sigma-Aldrich) diluted 100X was then added to 100 μl of the fixed parasite suspension. After 1 hr incubation at room temperature, the suspension was centrifuged, washed once and resuspended in 100 μl of PBS, before addition of the same volume of anti-mouse IgG-Alexa 488 (Thermo-Fisher Scientific) or Streptavidin-Phycoerythrin (BD Biosciences). The This website utilizes technologies such as cookies to enable essential site functionality, as well as for analytics, personalization, and targeted advertising. To learn more, view the following link: Privacy Policy samples were then incubated 30 min, washed, resuspended in 250 μl of PBS and fixed with the same volume of 4% p-formaldehyde in PBS. The samples were then analysed in Accury C6 flow cytometer (BD Biosciences).
7
Flow Cytometric Binding Assay for rPVL
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Cells were trypsinized, aliquoted, then washed twice in PBS (with Ca2+ and Mg2+). After incubation with rPVL-Alexa488 (5 and 10 μg ml-1), cells were washed twice and analysis was performed on an Accuri C6 flow cytometer using the CFlow-Plus Software (BD Biosciences). Alternatively, biotinylated rPVL or MAH were used, followed by Streptavidin-phycoerythrin (BD) and analysis was performed on a FACSCalibur with the Cellquest software (BD). Where indicated, cells were pre-teated with sialidase (Sigma) or ß-D-N-acetyl-hexosaminidase for 4h at 37°C.
8
Immunohistochemical Characterization of Tissues
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The following antibodies were used for immunohistochemical stainings of tissues: rabbit anti-mouse CD3 (1:100; Genetex, Hsinchu City, Taiwan; GTX25690), mouse anti-mouse CD4 (1:100; Abcam, Cambridge, UK; ab51312), rat anti-mouse CD31 (1:100; Dianova, Hamburg, Germany; DIA-310), rat anti-mouse CD45 (1:100; Abcam; ab25386), mouse anti-human C/EBPβ (1:100; Abcam; 18336) and rat anti-mouse MHCII (1:1000; eBioscience, San Diego, CA, USA; 14-5321).
For whole-mount immunohistochemistry, the following antibodies were used: biotinylated anti-mouse CD31 (1:100; BD Biosciences, San Jose, CA, USA; 553371), rabbit anti-mouse NG2 (1:100; Millipore, Billerica, MA, USA; AB5320), streptavidin-phycoerythrin (1:300; BD Biosciences; 554061) and swine anti-rabbit FITC (1:300; Dako; F0054).
For whole-mount immunohistochemistry, the following antibodies were used: biotinylated anti-mouse CD31 (1:100; BD Biosciences, San Jose, CA, USA; 553371), rabbit anti-mouse NG2 (1:100; Millipore, Billerica, MA, USA; AB5320), streptavidin-phycoerythrin (1:300; BD Biosciences; 554061) and swine anti-rabbit FITC (1:300; Dako; F0054).
9
Quantifying HIV-1 Antibody Subclasses
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Measurement of IgG subclass-specific HIV-1 binding Ab response was conducted as described above with some modifications. All plasma samples were tested against a limited panel of 5 HIV-1 antigens: B.con_env03 gp140, MNgp120, gp70_B.CaseA_V1V2, gp70 MNV3, and Rec MN gp41 at a 1:50 dilution. Antigen-specific subclass response was detected using biotinconjugated mouse anti-human IgG1 (BD Pharmingen, 4μg/L), IgG2 (Southern Biotech, 5ug/mL), IgG3 (Calbiochem, 2μg/mL), or IgG4 (BD Pharmingen, 2μg/mL) and tertiary detection agent streptavidin-phycoerythrin (BD Biosciences) at 5 μg/mL.
10
Flow Cytometric Analysis of Complement Activation
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Cells were washed with PBS and stained with anti-C5b-9 monoclonal antibody (Santa Cruz Biotechnology, Inc; dilution at 1/100) for 30 minutes on ice. Then, cells were washed with PBS and stained with Alexa 647–conjugated secondary antibody (1/500 dilution; Abcam) and Alexa 488–conjugated anti-C3c antibody (1/150 dilution; Abcam) for another 30 minutes on ice. The cells were also labeled with anti-C4d biotinylated monoclonal antibody (1/50 dilution; Quidel) and phycoerythrin-streptavidin (1/500 dilution; BD Pharmingen). Ten thousand events per sample were collected by a BD FACSCalibur and data were analyzed using FlowJo software version 10.5.3 (FlowJo Inc).
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