The adhesion rate was determined by dividing the number of mycobacteria bound to erythrocytes by the total number of mycobacteria inoculated in each well. We cocultured human erythrocytes with MAH organisms in medium supplemented with untreated or heat-treated serum for 1 h at 37°C, followed by washing erythrocytes with saline and centrifugation at 800 × g for 8 min at 4°C, three or four times. After washing, the erythrocytes were hemolyzed, and mycobacterial cell counts were assayed by determining the CFU. Erythrocytes were preincubated with 10 μg/mL of anti-human CR1 antibody or control antibody (IgGk) and complement components 3 or 2 for 30 min prior to the infection with MAH. For sialidase treatment, erythrocytes were incubated with 150 mU/mL sialidase (11080725001, Roche) at 37°C overnight, followed by washing the erythrocytes.
Sialidase
Manufactured by Roche
Sourced in United States, Germany, Switzerland
Sialidase is an enzyme that cleaves the glycosidic linkages of sialic acid residues from the non-reducing end of glycoproteins, glycolipids, and oligosaccharides. It is used in various laboratory applications for the analysis and modification of glycoconjugates.
Automatically generated - may contain errors
Lab products found in correlation
Pngase f, by Roche (5 mentions)
Sequencing grade trypsin, by Promega (4 mentions)
Formic acid, by Merck Group (3 mentions)
Asp n, by Roche (3 mentions)
Acetonitrile acn, by Biosolve (2 mentions)
Dithiothreitol (dtt), by Merck Group (2 mentions)
O glycosidase, by Roche (2 mentions)
Glu c, by Roche (2 mentions)
Trypsin, by G Biosciences (1 mentions)
Sialic acid quantification kit, by Merck Group (1 mentions)
Oseltamivir phosphate, by Merck Group (1 mentions)
Sambucus nigra lectin sna, by Vector Laboratories (1 mentions)
Siglec e sirna, by Santa Cruz Biotechnology (1 mentions)
Dynamo colorflash sybr green qpcr kit, by Thermo Fisher Scientific (1 mentions)
Giemsa stain, by Merck Group (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Reverse transcriptase kit, by Promega (1 mentions)
Rs504393, by R&D Systems (1 mentions)
Amicon ultra 0.5 centrifugal filter device, by Merck Group (1 mentions)
Alkaline phosphatase, by New England Biolabs (1 mentions)
20 protocols using sialidase
1
Mycobacterial Adhesion to Erythrocytes
2
Quantifying RBC-NP Glycoproteins and Sialic Acid
3
Oseltamivir and Sialidase Combination Therapy
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Oseltamivir Phosphate was obtained from Sigma-Aldrich. Oseltamivir was administered orally twice a day at 0.1 mg/g one day prior to infection [20 (link)]. Sialidase was purchased from Roche. Mice were treated with 0.012 U Sialidase in phosphate buffered saline (PBS) i.p. The treatment was performed twice a day, starting one day prior to infection. The treatment was adjusted from the published protocol [21 (link)].
4
Siglec-E Regulation in Macrophages
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Fluorescein isothiocyanate (FITC), bovine serum albumin (BSA), 4',6-diamidino-2-phenylindole (DAPI) and Giemsa stain were obtained from Sigma (St. Louis, MO). Sialidase was from Roche Applied Science (Mannheim, Germany); Mounting medium was from Amersham Biosciences (Uppsala, Sweden); Sambucus Nigra lectin (SNA) and Maackia amurensis lectin (MAL) were from Vector Labs; 2'7'- dichloro dihydro fluorescein diacetate acetyl ester (H2DCFDA) was from Molecular probes (OR, USA); siglec-E siRNA was from santa cruz Biotechnology and DyNAmo ColorFlash SYBR Green qPCR kit was from Thermo Scientific (USA). All the cytokine ELISA kits, PE-rat anti-mouse CD14 and SHP-1 antibody were obtained from BD pharmingen and BD Biosciences (USA). Anti-Siglec-E antibody was from R&D systems (MN, USA); RNeasy Mini Kit was from Qiagen (Limburg, Netherlands); Reverse Transcriptase Kit was from Promega (WI, USA) and PCR reagent system was from Invitrogen (CA, USA). All other antibodies were from Cell Signaling Technologies (MA, USA), unless indicated otherwise.
5
Sialidase Treatment for Lectin Analysis
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Single cells were resuspended in sodium citrate buffer (pH 6) and treated with 0.5 units/ml sialidase from C. perfringens (Roche) in sodium citrate buffer (pH 6) for 1 h at 37°C. After treatment, cells were washed with serum-free medium or cold PBS, for cell attachment assays or flow cytometry, respectively. For the specificity of the lectins in the lectin blot analysis, part of the membrane was treated with sialidase for 16 h at 37°C.
6
Purification and Analysis of Complement C8
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Complement component C8 (UniProt Code: P07357 (α), P07358 (β), P07360 (γ)) purified from pooled human blood plasma (several healthy donors) was acquired from Complement Technology, Inc. (Texas, USA). The sample was purified according to a reported standard protocol [18 (link)] (the certificate of analysis is attached in the Supporting information – S1). Dithiothreitol (DTT), iodoacetamide (IAA) and ammonium acetate (AMAC) were purchased from Sigma-Aldrich (Steinheim, Germany). Formic acid (FA) was from Merck (Darmstadt, Germany). Acetonitrile (ACN) was purchased from Biosolve (Valkenswaard, The Netherlands). POROS Oligo R3 50-μm particles were obtained from PerSeptive Biosystems (Framingham, MA, USA) and packed into GELoader pipette tips (Eppendorf, Hamburg, Germany). Sequencing grade trypsin was obtained from Promega (Madison, WI). Asp-N, PNGase F, and Sialidase were obtained from Roche (Indianapolis, USA).
7
Anti-inflammatory Effects of sSiglec-9
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To determine whether sialic acid and C-C chemokine receptor type 2 (CCR2) were required to induce the anti-inflammatory effects of sSiglec-9, we used sialidase [23 (link)] (Roche, Basel, Switzerland) to remove sialic acid and RS504393 (R&D Systems), a selective CCR2 antagonist, to inhibit the CCR2/CCL2 signaling pathway [24 ]. RAW264.7 cells were preincubated with 15 mU/300 μl sialidase for 1 h and 50 μM RS504393 for 20 minutes before stimulation.
8
Desialylation of Mouse Transferrin
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To study the effects of sialylation on glycoprotein structure, fully sialylated mouse transferrin was de-sialylated by using Sialidase of neuraminidase under native conditions. Neuraminidase (Sialidase, from C. Perfringens 11585886001 Roche) was supplied by Sigma-Aldrich (St. Louis, MO). First, we dissolved/reconstituted the enzyme to 5 mL water (Sialidase concentration: 1 U/mL). Then, a volume of 100 μL glycoprotein (mouse transferrin, 1 mg mL−1 in 100 mM NH4OAc, pH 7) was added to a 600 μL EP tube. Then 25 μL Sialidase (1 U/mL) was added, while for the control group, 25 μL NaOAc (0.1 M, pH 5) was added instead. After vortex mixing, parafilm sealing, the mixtures were incubated in water bath at 37 °C for 5 h. The de-sialylation was stopped with the addition of 25 μL NaHCO3 (0.5 M). After de-sialylation, proteins were buffer-exchanged into 100 mM NH4OAc with Amicon Ultra-0.5 Centrifugal Filter Devices (30 K MWCO: UFC503024, Sigma-Aldrich (St. Louis, MO)) following supplier’s standard procedures.
9
Sialidase Treatment Impacts ShaV Infection
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MARC-145 cells were treated with sialidase (Roche, Switzerland) from Arthrobacter ureafaciens (10 U/mL), Vibrio cholerae (1 U/mL), and Clostridium perfringens (5 U/mL), following a 2-fold serial dilution from 500 mU/mL in DMEM. Prior to ShaV infection, cell monolayers in 96-well plates were incubated with 100 μL of diluted sialidases at the indicated concentrations for 1 h at 37°C. After washing with Dulbecco’s phosphate-buffered saline (DPBS), cells were inoculated with ShaV at an MOI of 0.1 or 1 for 1 h at 37°C. The mock controls were treated with DMEM only. The cells were then washed with DPBS and maintained in DMEM (2.5% FBS) for 12 h. The experiments were repeated six times (n = 6).
10
Sialidase Treatment Impacts ShaV Infection
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MARC-145 cells were treated with sialidase (Roche, Switzerland) from Arthrobacter ureafaciens (10 U/mL), Vibrio cholerae (1 U/mL), and Clostridium perfringens (5 U/mL), following a 2-fold serial dilution from 500 mU/mL in DMEM. Prior to ShaV infection, cell monolayers in 96-well plates were incubated with 100 μL of diluted sialidases at the indicated concentrations for 1 h at 37°C. After washing with Dulbecco’s phosphate-buffered saline (DPBS), cells were inoculated with ShaV at an MOI of 0.1 or 1 for 1 h at 37°C. The mock controls were treated with DMEM only. The cells were then washed with DPBS and maintained in DMEM (2.5% FBS) for 12 h. The experiments were repeated six times (n = 6).
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