Organs were collected from WT, CD45.1, CD45.2, OT-I, or CD8β-deficient mice and single-cell suspensions prepared using standard protocols. Following removal of red blood cells using ACK, nonspecific receptors were blocked with monoclonal antibody (mAb) 2.4G2, before cells (1–5 × 106) were stained with mAb to mouse NKp46 (29A1.4; BioLegend), CD3 (17A2; BD Biosciences), TCRγδ (GL3, BioLegend), TCRαβ (H57-597, BioLegend), CD8α (53-6.7; BD Biosciences), CD8β (H35-17.2; BD Biosciences). Alternatively cells were stained with the lectins peanut agglutinin (Vector Laboratories), sambucus nigra lectin (Vector Laboratories), or maackia amurensis lectin II (Vector Laboratories) before detecting using Streptavidin (BD Biosciences). Cells stained with tetramers were fixed in 2% paraformaldehyde for 15 min and washed twice with FACS buffer (1% FCS/PBS) before being resuspended in FACS buffer. All other FACS combinations were acquired unfixed. For acquisition, events were electronically gated on FSC-A versus FSC-H (singlets), followed by FSC-A and SSC-A (to exclude doublets and debris). Among the remaining population, at least 5000 electronic events of interest were collected using an LSR-II or X- 20 Fortessa (BD Biosciences).
Maackia amurensis lectin 2
Manufactured by Vector Laboratories
Sourced in United States
Maackia amurensis lectin II is a plant-derived protein that exhibits specific binding to sialic acid residues. It is commonly used in glycobiology research applications as a tool for the detection and analysis of sialic acid-containing glycoconjugates.
Automatically generated - may contain errors
Lab products found in correlation
Sambucus nigra lectin, by Vector Laboratories (3 mentions)
Fortessa x20, by BD (1 mentions)
Peanut agglutinin, by Vector Laboratories (1 mentions)
Cd8α 53 6.7, by BD (1 mentions)
Cd3 17a2, by BD (1 mentions)
Tcrγδ gl3, by BioLegend (1 mentions)
Facscanto 1, by BD (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Wheat germ agglutinin (wga), by Vector Laboratories (1 mentions)
Jacalin, by Vector Laboratories (1 mentions)
Ez ecl substrate, by Sartorius (1 mentions)
Ecl chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Novex bis tris gel, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Elderberry bark lectin, by Vector Laboratories (1 mentions)
Trans blot sd semi dry transfer cell, by Bio-Rad (1 mentions)
Las 4000, by Cytiva (1 mentions)
6 protocols using maackia amurensis lectin 2
1
Multiparameter Flow Cytometry Analysis of Immune Cell Populations
2
Sialic Acid Levels in CLL Leukocytes
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Leukocytes from PB of CLL patients were isolated using an ammonium chloride-based red blood lysis buffer (155 mM ammonium chloride, 10 mM potassium bicarbonate, and 0.2 mM ethylenediaminetetraacetic acid [EDTA] tetrasodium salt, all from Merck; Rahway, NJ, USA). To determine the levels of α2-3 and α2-6 linked sialic acids, leukocytes (2x106 per tube) were incubated for 15 minutes (min) at room temperature (RT) with gentle rocking with Maackia amurensis lectin II and Sambucus nigra lectin (1:20000; Vector Laboratories; Newark, CA, USA), which preferentially bind the α2-3 and α2-6 linked sialic acids, respectively. After incubation, cells were washed, stained with APC-conjugated streptavidin beads (1:400; BD Bioscience, Franklin Lakes, NJ, USA), FITC-conjugated anti-CD5 and PE-conjugated anti-CD19 antibodies (both from Immunological Science, Rome, Italy), AlexaFluor 647-conjugated cutaneous lymphocyte antigen antibody (clone Heca452; BD Bioscience) and incubated for 30 min at RT with gentle rocking. After incubation, cells were washed, resuspended in 500 µL staining buffer supplemented with 7-aminoactinomycin D (7-AAD, 1:80; Immunological Science) to exclude dead cells. Cells were acquired on a BD FACS Canto I (BD Biosciences). Data were analyzed using the Infinicyt software v 2.0.5.b.007 (Cytognos; Salamanca, Spain).
3
H9N2 Virus Infection and PD-L1 Induction
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The H9N2 virus (Ck/HB/4/08) was inoculated into 9-day-old SPF chicken embryos, and virus titers were determined by measuring PFUs. Madin-Darby canine kidney (MDCK, CCL-34, ATCC) cells used for PFUs were cultured in DMEM media supplemented with 5% FBS. AIV-specific sialic acid α-2,3-galactose receptor (SA2-3Gal) expression was confirmed by biotinylated Maackia amurensis lectin II (VECTOR, CA, USA) staining and then followed by staining with FITC-conjugated avidin D (green) and DAPI (blue) for nuclei. To assess H9N2 virus infection, RPMECs were washed with PBS, inoculated with virus at different multiplicities of infection (MOIs) and incubated for 1 h. Then, the cells were washed with PBS and incubated with DMEM, 0.2% bovine serum albumin (Gibco, Carlsbad, CA, USA) and 0.2 μg/mL TPCK-treated trypsin [20 (link)]. Viral titers in the supernatants were measured using PFUs. To investigate the PD-L1 level induced by inactivated H9N2 virus, viral particles were inactivated using 0.094% β-propionolactone (BPL; SERVA Electrophoresis, Heidelberg, Germany) according to a previously described protocol [21 (link)].
4
Immunoprecipitation and Lectin Blotting of Ncr1
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Immunoprecipitation of Ncr1 was performed following standard immunoprecipitation protocol, using agarose A/G beads and the anti-Ncr1 mNcr1.6 mAb. For western blotting, the precipitated proteins were run on a 10% SDS-PAGE gel, transferred and stained with anti-mNcr1. For the western blotting with lectins, the various fusion proteins (5 μg) were run on 10% SDS-PAGE gel, transferred to a nitrocellulose membrane (Tamar, Mevaseret Zion, Israel) and blotted with biotin-conjugated lectins (Jacalin, wheat germ agglutinin, Maackia amurensis lectin II and SNA, all from Vector labs, Burlingame, CA, USA) or with biotin-conjugated HA Ig. The staining was visualized using a streptavidin HRP secondary reagent and EZ ECL substrate (Biological Industries, Beit-Haemek, Israel).
5
Glycosylation Analysis of LDL Proteins
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LDL samples were subjected to NuPAGE using 3% to 8% Novex Bis‐Tris gels (Invitrogen) and transferred to nitrocellulose membrane. Blots were blocked with 50 mmol/L Tris‐HCl, pH 7.4, containing 150 mmol/L NaCl, 3% (w/v) BSA, and 0.05% Tween 20 at room temperature for 1 hour, and then incubated overnight with biotinylated Maackia amurensis lectin‐II, Sambucus nigra lectin, or peanut (Arachis hypogaea) agglutinin (Vector Laboratories) in the same buffer containing 1% BSA at 4°C. After washing with Tris‐buffered saline–Tween (0.05% v/v), blots were incubated with horseradish peroxidase–conjugated streptavidin for 1 hour at room temperature. Following washing with Tris‐buffered saline–Tween, blots were developed using ECL chemiluminescence reagent (Thermo Fisher Scientific) and analyzed using x‐ray film. Ponceau S–stained membranes were used as a control for equal protein loading.
6
Glycosylation Pattern Analysis of Antibodies
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To determine the glycosylation pattern, 1 μg/mL reduced Ab was loaded on a 10% SDS-PAGE gel. After separation, proteins were transferred to a nitrocellulose membrane (GE Healthcare, Amersham, UK) using a semi-dry blotting system (Bio-Rad Trans-Blot SD Semi-Dry Transfer Cell, Hercules, CA, USA). Membranes were then blocked with a casein containing western blocking reagent (Roche, 11921673001, Mannheim, Germany) in Tris-buffered saline (TBS; 50 mM Tris-Cl in ddH2O [pH 7.6]) overnight. Blots were washed twice with TBS + 0.1% Tween 20 and incubated with a 1:1,000 dilution of biotinylated Maackia Amurensis Lectin II (Vector Laboratories; binds α2-3 sialic acid [SA]) in TBS + 0.1% Tween 20 or a 1:1,000 dilution of biotinylated Elderberry Bark Lectin (Vector Laboratories; binds α2-6 SA) in TBS + 0.1 mM CaCl2, MgCl2, and MnCl2 + 0.1% western blot (WB) blocking reagent + 0.1% Tween 20. After washing six times with TBS + 0.1% Tween 20 for 5 min, streptavidin-horseradish peroxidase (HRP) diluted 1:2,000 in TBS was added and incubated for 30 min. Membranes were washed again six times with TBS + 0.1% Tween 20 for 5 min and rinsed once with TBS. Membranes were developed using chemiluminiscent enhanced chemiluminescence (ECL) substrate and a LAS 4000 Mini chemiluminescent image analyzer (ImageQuant, GE Healthcare, Amersham, UK).
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