Labeled and unlabeled ricin toxin (Ricinus communis agglutinin II) and Ricinus communis agglutinin I (RCA-I) were purchased from Vector Laboratories (Burlingame, CA, USA). Ricin was dialyzed against phosphate buffered saline (PBS) at 4 °C in 10,000 MW cutoff Slide-A-Lyzer dialysis cassettes (ThermoFisher Pierce Protein Biology, Rockford, IL, USA) prior to use in cytotoxicity studies. Unless noted otherwise, all of the other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA). Vero cells and THP-1 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and propagated as recommended by ATCC. Cell culture media were provided by the Wadsworth Center’s tissue culture facility.
Ricinus communis agglutinin 1 rca 1
Manufactured by Vector Laboratories
Sourced in United States
Ricinus communis agglutinin I (RCA I) is a lectin purified from the seeds of the castor bean plant (Ricinus communis). It is a carbohydrate-binding protein that specifically recognizes and binds to galactose-containing glycoconjugates. RCA I is often used as a tool in biochemical and cell biology research applications.
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4 protocols using ricinus communis agglutinin 1 rca 1
1
Ricin Toxin and RCA-I Cytotoxicity Assay
2
Evaluation of Ricinus communis Agglutinin I
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All of the reagents used were analytical-grade and, unless specified, all of the other reagents and chemicals were purchased from Sigma-Aldrich. Bovine serum albumin (BSA; 66.5 kDa and ∼96%), D-lactose monohydrate (Lac), glutaraldehyde (25%), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin (P/S), fluorescein isothiocyanate (FITC), and [3-5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ricinus communis agglutinin I (RCA I) was obtained from the Vector Lab (Burlingame, CA, USA). HepG2 (human liver cancer) and HeLa (human cervical carcinoma) cells were obtained from ATCC (American Type Culture Collection, Manassas, VA, USA).
3
Synthesis and Evaluation of Lactose-Conjugated Doxorubicin
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All of the reagents used were analytical grade. Bovine serum albumin (BSA; 66.5 kDa and ~96%), D-lactose monohydrate (Lac), glutaraldehyde (25%), doxorubicin (Dox) hydrochloride, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin (P/S), and [3-5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ricinus communis agglutinin I (RCA I) was obtained from the Vector Lab (Burlingame, CA, USA). Human liver cancer cells (HepG2 cells) and human cervical carcinoma cells (HeLa cells) were obtained from ATCC (Manassas, VA, USA). Unless specified, all other reagents and chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). All the experiments were carried out using Type 2 pure water (0.18 μS cm−1).
4
Platelets Desialylation and Lectin Binding
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Desialylation of platelets was confirmed by lectin binding, using flow cytometric analysis. Fluorescein isothiocyanate (FITC)-conjugated Ricinus Communis agglutinin I (RCA-I; Vector Laboratories, Burlingame, CA, USA) was used for assessing platelet surface β-GlcNac exposure [8 (link), 13 (link)]. RCA-I at 5.0 µg/mL was added to each of isolated platelets after resuspension in buffer B. In the positive control group, 0.3 U/mL of sialidase was also added. The groups of platelets stored at 4 °C were then incubated at 37 °C for 20 min, using a WiseTherm® HB-R (Daihan Sci., Seoul, Korea) water bath, and the groups of platelets stored at 22 °C were incubated with continuous gentle agitation. The lectin binding of platelets was analyzed using a Beckman Navios flow cytometer (Beckman Coulter Inc., Brea, CA, USA). Platelets were gated according to their forward scatter (FSC) and side scatter (SSC) characteristics.
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