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Np osu

Manufactured by Biosearch Technologies
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Sourced in Sao Tome and Principe, United States

The NP-Osu is a laboratory equipment designed for the purification and analysis of nucleic acids. It utilizes magnetic nanoparticles to capture and separate various nucleic acid species from complex samples. The core function of the NP-Osu is to provide a reliable and efficient method for nucleic acid extraction and purification in research and diagnostic applications.

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Lab products found in correlation

Succinimide ester, by Biosearch Technologies (2 mentions) Bl21 de3 competent cells, by Thermo Fisher Scientific (2 mentions) Phycoerythrin, by Merck Group (1 mentions) Np klh, by Merck Group (1 mentions) Keyhole limpet hemocyanin (klh), by Merck Group (1 mentions) Streptavidin alexa 488, by Thermo Fisher Scientific (1 mentions) Icosl, by BioLegend (1 mentions) Streptavidin alkaline phosphatase, by Southern Biotech (1 mentions) Anti igg alkaline phosphatase, by Southern Biotech (1 mentions) Multiscreen htsip filter plates, by Merck Group (1 mentions) Instantblue ultrafast protein stain, by Merck Group (1 mentions) Fitc dextran, by Merck Group (1 mentions) Np2 bsa, by Biosearch Technologies (1 mentions) Anti igd 11 26c 2a, by BioLegend (1 mentions) Anti gfp antibody, by Abcam (1 mentions) Anti fas jo2, by BD (1 mentions) Np pe, by Biosearch Technologies (1 mentions) Np18 ova, by Biosearch Technologies (1 mentions) Dextran, by Merck Group (1 mentions)

9 protocols using np osu

1

Synthesis of NP-Streptavidin Conjugates

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4-Hydroxy-3-nitrophenylacetic acid Succinimide Ester (NP-Osu, Biosearch Technologies) was conjugated to Alexa Fluor 647 Streptavidin (SA-AF647) or Alexa Fluor 594 Streptavidin at a hapten:streptavidin molar ratio of 10:1 or 20:1. Biotinylated Eα52–73 peptide (N-biotin-GSGFAKFASFEAQGALANIAVDKA-COOH)15 (link),77 (link) was synthesized at the RU Proteomics Resource Center. NP-Streptavidin conjugates were incubated with a 6x molar excess of biotinylated Eα peptide, and excess peptide was removed by dialysis. Hapten-protein conjugation ratios were calculated by measuring the absorbance value at 430 nm. For αDEC-OVA-Eα experiments shown in Figures S4E and S4F, NP-SA-AF647 conjugates were incubated with a 30x molar excess of D-biotin and excess D-biotin was removed by dialysis.
Chen S.T., Oliveira T.Y., Gazumyan A., Cipolla M, & Nussenzweig M.C. (2023). B cell receptor signaling in germinal centers prolongs survival and primes B cells for selection. Immunity, 56(3), 547-561.e7.
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2

Synthesis of NP-Streptavidin Conjugates

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4-Hydroxy-3-nitrophenylacetic acid Succinimide Ester (NP-Osu, Biosearch Technologies) was conjugated to Alexa Fluor 647 Streptavidin (SA-AF647) or Alexa Fluor 594 Streptavidin at a hapten:streptavidin molar ratio of 10:1 or 20:1. Biotinylated Eα52–73 peptide (N-biotin-GSGFAKFASFEAQGALANIAVDKA-COOH)15 (link),77 (link) was synthesized at the RU Proteomics Resource Center. NP-Streptavidin conjugates were incubated with a 6x molar excess of biotinylated Eα peptide, and excess peptide was removed by dialysis. Hapten-protein conjugation ratios were calculated by measuring the absorbance value at 430 nm. For αDEC-OVA-Eα experiments shown in Figures S4E and S4F, NP-SA-AF647 conjugates were incubated with a 30x molar excess of D-biotin and excess D-biotin was removed by dialysis.
Otto-Bruc A.E., Fariss R.N., Van Hooser J.P, & Palczewski K. (1998). Phosphorylation of photolyzed rhodopsin is calcium-insensitive in retina permeabilized by α-toxin. Proceedings of the National Academy of Sciences of the United States of America, 95(25), 15014-15019.
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3

Adoptive Transfer of B Cells in μMT Mice

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For the adoptive transfer of B cells into μMT mice, splenic B cells from naïve WT or BAFFR-/- were isolated by negative selection enrichment (STEMCELL Technologies, Vancouver, BC, Canada). Highly purified B cells were obtained by both WT and BAFFR-/- mice, as 98% of B cells purified from naïve BAFFR-/- mice were T1 B cells. 2 × 107 purified total B cells from WT mice and T1 B cells from BAFFR-/- mice or PBS as vehicle control were transferred i.v. into μMT recipients 18 h before immunization. The hapten 4-hydroxy-3-nitro-phenacetyl (NP) was conjugated to anti-CD180 (NP-αCD180) as previously reported [27 (link)]. The adoptively transferred mice were inoculated i.v. with 200 μl of PBS containing 100 μg/mouse of NP-αCD180. For direct immunizations of WT and BAFFR-/- mice, mice were inoculated i.v. with 50 μg/mouse of NP-αCD180. ELISA and ELISPOT assays to detect NP-IgG in the serum and NP-IgG ASCs in the spleens were performed as previously described [27 (link)]. To detect NP-specific cell subsets by flow cytometry NP-PE was prepared by conjugation of NPOsu (Biosearch Technologies) to phycoerythrin (Sigma-Aldrich, St. Louis, MO) as described [27 (link)].
Giordano D., Draves K.E., Young L.B., Roe K., Bryan M.A., Dresch C., Richner J.M., Diamond M.S., Gale M J.r, & Clark E.A. (2017). Protection of mice deficient in mature B cells from West Nile virus infection by passive and active immunization. PLoS Pathogens, 13(11), e1006743.
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4

Synthetic GPI and NP-KLH Conjugation

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Synthetic glycosylphosphatidylinositol (GPI) was conjugated to maleimide activated Keyhole Limpet Haemocyanin (KLH) using 2-iminothiolane and stored at −80°C until use (GPI-KLH). Four-hydroxy-3-nitrophenyl acetyl-Osu (NP-Osu) (Biosearch Technologies, USA) was conjugated to KLH (Sigma-Aldrich, USA; molar ratio between 13 and 20) according to manufacturer's instructions and stored at −20°C until use (NP-KLH).
Joseph H., Tan Q.Y., Mazhari R., Eriksson E.M, & Schofield L. (2019). Vaccine-Induced Carbohydrate-Specific Memory B Cells Reactivate During Rodent Malaria Infection. Frontiers in Immunology, 10, 1840.
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5

Antibody Reagents for B Cell Immunology

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Antibodies against mouse CD4, CD19, CD95, GL-7, ICOS, ICOSL, PD-1, and B220-647 were purchased from Biolegend, CXCR5, B220, IgG1, IgG2a, IgG2b, and IgG3 antibodies from BD Biosciences, Bcl-6, XBP-1, and IRF-4 from Santa Cruz, and BAFF (1C9) from Enzo. Anti-μ F(ab)2 was purchased from Jackson ImmunoResearch. Anti-μ (clone B7.6), anti-NP (clones H33L and B1-8), 2.4G2 (FcγRIIb /FcγRIII block) and Ac38 idiotype antibodies were purified from hybridoma supernatants. (Ac38 is an idiotype antibody that recognizes B-1-8 specificities generated during NP immunization). Recombinant mouse BR3-Fc and isotype control protein were generated using mammalian expression systems and standard purification protocols. H33L and B1-8 were gifts from Dr. Garnett Kelsoe (Duke University). IL-4 and IL-5 were purchased from Peprotech, recombinant BAFF (rBAFF) from R&D Systems, NP-OSu from Biosearch Technologies, KLH and PNA-biotin from Sigma Aldrich, alum from Thermo Scientific, and streptavidin-Alexa 488 and Alexa 647 from Invitrogen. Streptavidin-alkaline phosphatase and anti-IgG alkaline phosphatase were purchased from Southern Biotech. The Fc-binding TG19320 peptide was synthesized as described (39 (link), 40 (link)).
Kang S., Keener A.B., Jones S.Z., Benschop R.J., Caro-Maldonado A., Rathmell J.C., Clarke S.H., Matsushima G.K., Whitmire J.K, & Vilen B.J. (2015). IgG-immune complexes promote B cell memory by inducing BAFF. Journal of immunology (Baltimore, Md. : 1950), 196(1), 196-206.
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6

ELISA and ELISPOT Protocols for Protein Analysis

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Enzyme-linked immunosorbent assays (ELISAs) were done as described (48 (link)) with addition of 0.05% Tween 20 in block and wash buffers. 4-Hydroxy-3-nitrophenylacetyl hapten (NP) conjugated to BSA (NP-BSA, Ratio 28) was generated in-house with BSA fraction V (Roth Cat# 8076.3) and NP-OSu (Biosearch Technologies Cat# N1010-100). Plates were coated with 2 µg/ml NP-BSA or 1 µg/ml anti-light chain antibodies and developed with 1 µg/ml anti-isotype antibodies and the standards listed in Supplementary Table 1. For enzyme-linked immuno spot (ELISPOT) assays MultiScreenHTS IP Filter Plates (Merck Cat# MSIPS4510) were coated and developed as described above for the ELISA plates, incubated with cells overnight, washed with 0.1% Tween 20 and processed according to the manufacturer’s instructions. For serum protein electrophoresis or immunofixation 10 µl serum was run on buffered agarose gels, pH8.6 Hydragel PROTEIN(E) (Sebia Cat# PN4100) or pH9.2 DOUBLE IF K20 (Sebia Cat# PN3036), and processed according to the manufacturer’s instructions. For proteomics, serum samples were run on multiple lanes of pH8.6 agarose gels and stained with InstantBlue Ultrafast Protein Stain (Sigma Cat# ISB1L). Excised bands were processed and analyzed by tandem mass spectrometry as described below.
Schmidt K., Sack U., Graf R., Winkler W., Popp O., Mertins P., Sommermann T., Kocks C, & Rajewsky K. (2020). B-Cell-Specific Myd88 L252P Expression Causes a Premalignant Gammopathy Resembling IgM MGUS. Frontiers in Immunology, 11, 602868.
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7

Murine B Cell Phenotyping and Detection

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Fluorophore-labeled anti-B220 (RA3-6B2) and anti-FAS (Jo2) antibodies were from BD; anti-CD138 (281-2), anti-CD21/35 (CR2/CR1), and anti-IgD (11-26c2a) antibodies were from BioLegend; anti-GL7 (GL7), anti-CD38 (90), and anti-rabbit IgG antibodies were from Invitrogen; rabbit anti-cCasp3 and the matching isotype control antibodies were from Cell Signaling; and anti-GFP antibody was from Abcam. The fixable viability dye, zombie yellow, was from BioLegend. NP18-OVA, NP30-Ficoll, NP2-BSA, NP-PE, and 4-hydroxy-3-iodo-5-nitrophenylacetyl (NIP)-BSA-biotin were from Biosearch Technologies. NP-APC conjugates were made by allowing NP-Osu (Biosearch Technologies) and APC (BioLegend) to react for 4 h at room temperature in a buffer containing 0.1 M NaHCO3 and 0.15 M NaCl2 (pH 8), at a molar ratio of 20:1. Dextran (200 kD, 31398) and Dextran-FITC (2,000 kD, FD2000S) were from Sigma-Aldrich.
Liu X., Zhao Y, & Qi H. (2022). T-independent antigen induces humoral memory through germinal centers. The Journal of Experimental Medicine, 219(3), e20210527.
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8

Production and Characterization of LLO(LT) Conjugates

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All peptides used in this study were purchased from Peptide 2.0 Inc., purified by reverse-phase high pressure liquid chromatography, and analyzed by mass-spectroscopy. A pET29b expression vector containing the His-tagged LLOLT sequence was provided by Dr. Emil Unanue (Washington University, St. Louis, MO)36 (link). For generation of LLOLT-N, site-directed mutagenesis was used to change the lysine at position 203 to an asparagine (N). Mutated clones were confirmed by sequencing. LLOLT and LLOLT-N were expressed in BL21 (DE3) competent cells (ThermoFisher) and purified as previously described36 (link). Protein purity was confirmed by SDS-PAGE. For conjugation of NP to LLOLT and LLOLT-N, 0.5mg of NP-OSu (LGC Biosearch Technologies) was added to 5mg of either LLOLT or LLOLT-N in 10 equal fractions over 20 minutes. The solution was then incubated at room temperature with rotations for 2 hours before being dialyzed into 0.1M NaHCO3, 145mM NaCl, pH 8.5. NP:LLOLT(-N) ratio was determined with the following extinction coefficients: LLOLT(-N) = 75750 M−1cm−1 and NP = 4230 M−1cm−1. Ratios of NP:LLOLT(-N) ranged from 5:1-10:1, and batches of NP-LLOLT(-N) were aliquoted and stored at -20°C to allow for continuity across experiments.
Bartleson J.M., Milam A.A., Donermeyer D.L., Horvath S., Xia Y., Egawa T, & Allen P.M. (2020). Strength of tonic T cell receptor signaling instructs T follicular helper fate decisions. Nature immunology, 21(11), 1384-1396.
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9

Production and Characterization of LLO(LT) Conjugates

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All peptides used in this study were purchased from Peptide 2.0 Inc., purified by reverse-phase high pressure liquid chromatography, and analyzed by mass-spectroscopy. A pET29b expression vector containing the His-tagged LLOLT sequence was provided by Dr. Emil Unanue (Washington University, St. Louis, MO)36 (link). For generation of LLOLT-N, site-directed mutagenesis was used to change the lysine at position 203 to an asparagine (N). Mutated clones were confirmed by sequencing. LLOLT and LLOLT-N were expressed in BL21 (DE3) competent cells (ThermoFisher) and purified as previously described36 (link). Protein purity was confirmed by SDS-PAGE. For conjugation of NP to LLOLT and LLOLT-N, 0.5mg of NP-OSu (LGC Biosearch Technologies) was added to 5mg of either LLOLT or LLOLT-N in 10 equal fractions over 20 minutes. The solution was then incubated at room temperature with rotations for 2 hours before being dialyzed into 0.1M NaHCO3, 145mM NaCl, pH 8.5. NP:LLOLT(-N) ratio was determined with the following extinction coefficients: LLOLT(-N) = 75750 M−1cm−1 and NP = 4230 M−1cm−1. Ratios of NP:LLOLT(-N) ranged from 5:1-10:1, and batches of NP-LLOLT(-N) were aliquoted and stored at -20°C to allow for continuity across experiments.
Bartleson J.M., Milam A.A., Donermeyer D.L., Horvath S., Xia Y., Egawa T, & Allen P.M. (2020). Strength of tonic T cell receptor signaling instructs T follicular helper fate decisions. Nature immunology, 21(11), 1384-1396.
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