Concanavalin a (cona)

Manufactured by Thermo Fisher Scientific

Sourced in United States

Concanavalin A is a type of lectin protein derived from the jack bean (Canavalia ensiformis). It has the ability to agglutinate (clump) certain types of cells, including red blood cells and lymphocytes. Concanavalin A is commonly used as a research tool in cell biology and biochemistry applications.

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68 protocols using concanavalin a (cona)

Evaluating T-Cell Activation in Mouse Immune Cells

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Mouse mononuclear cells were treated with ConA (2 μM, Thermo Fisher Scientific), ConA plus EV^GLN− preparations (10 μg/ml), or EV^GLN+ preparations (10 μg/ml) for 72 hours (n = 3). After treatment, cells from each group were collected and stained with fluorescein isothiocyanate (FITC)–conjugated anti-mouse CD3e (553061, BD, Brea, CA, USA, USA), peridinin chlorophyll protein (PerCP)/Cyanine5.5-conjugated anti-mouse CD4 (100434, BioLegend), phycoerythrin (PE)–Cy7–conjugated anti-mouse CD8a (552887, BD), and allophycocyanin (APC)–conjugated anti-mouse CD69 (APC-65105, Proteintech, Wuhan, China) for 30 min. After washing with PBS, the stained cells were analyzed using a flow cytometer (LSRFortessa, BD Biosciences).

Wang Y., Lou P., Xie Y., Liu S., Li L., Wang C., Du D., Chen Y., Lu Y., Cheng J, & Liu J. (2024). Nutrient availability regulates the secretion and function of immune cell–derived extracellular vesicles through metabolic rewiring. Science Advances, 10(7), eadj1290.

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Ex vivo Reactivation Assay for FIV

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To determine if the tissue-derived leukocytes contained replication-competent virus, an ex vivo reactivation assay was performed as previously described for PBMCs isolated from FIV-infected cats with the following modifications.[11 ,12 (link)] Approximately 5 x 10⁶ MLN or splenic-derived leukocytes were cultured ex vivo with allogeneic SPF PBMCs (2.5 x 10⁶) in RPMI media (Hyclone) supplemented with 100 units/ml human IL-2 (NIH AIDS Reagent Program) and 5μg/ml of mitogen concanavalin-A (Con-A; ThermoFisher Scientific). Due to reduced numbers of total leukocytes collected from cat 184 (from both MLN and spleen) only 3 and 4.25 x 10⁶ leukocytes were used in the reactivation assay for MLN and spleen respectively. Cultures were passaged as previously described and clarified cell-free supernatants were collected on days 7, 14, and 21 for cryopreservation.[11 ] At a later time, collected supernatants were independently passaged onto approximately 7 x 10⁶ Con-A activated allogeneic SPF PBMC. Cultured allogeneic cells were subsequently assayed for the presence of gag DNA (provirus) by real-time PCR seven days after inoculation with clarified supernatant. Detectable vDNA was interpreted to be consistent with successful reactivation from tissue leukocytes of infected cats. Fresh MLN and spleen leukocytes from all four FIV positive cats, and one negative control cat (185) were assayed.

Eckstrand C.D., Sparger E.E., Pitt K.A, & Murphy B.G. (2017). Peripheral and central immune cell reservoirs in tissues from asymptomatic cats chronically infected with feline immunodeficiency virus. PLoS ONE, 12(4), e0175327.

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Redirecting T cells for CD20+ cell killing

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Effector cells were generated by stimulating human CD3 transgenic T cells in vitro with ConA (ThermoFisher, 00-4978-03) for 2 days, and then resting the cells for 7 days. The effector cells were then labeled with CellTrace Violet (ThermoFisher Scientific, C34571). Target cells expressing human CD20 were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; C34554, ThermoFisher) and aliquoted to V-bottom 96-well plates at a density of 2 × 10⁴ cells per well. Effector cells were added at a density of 4 × 10⁴ cells per well to achieve an effector-to-target ratio of 2:1, and total volume were adjusted to 200 μL. The plates were centrifuged at 200g for 3 minutes and cultured at 37°C with 5% CO₂. CD20 or CD123 BsAbs were added to a final concentration of 50 ng/mL. After 5 hours, cells were harvested and stained with annexin-V/propidium iodide to evaluate the viability/killing of target cells. CountBright Absolute Counting Beads (ThermoFisher Scientific) were added to estimate the absolute number of cells by flow cytometry. Target and effector cells were distinguished by prelabeled live cell dye (CFSE for target cells and CellTrace Violet for effector cells).

Wang L., Leach V., Muthusamy N., Byrd J, & Long M. (2023). A CD3 humanized mouse model unmasked unique features of T-cell responses to bispecific antibody treatment. Blood Advances, 8(2), 470-481.

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Mammalian Cell Culture Maintenance Protocol

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Mammalian cells were maintained in T75 flasks and subcultured every 3–4 days. Complete growth media contained 10% FBS and 1% antibiotic cocktail. CTLL-2 cells (RRID:CVCL_0227) were cultured in complete RPMi media supplemented with 10% T-cell culture supplement with ConA (Thermofisher Scientific 11513540). CT-26 cells (RRID:CVCL_7254) were cultured in complete RPMi media. HT29 cells (RRID:CVCL_A8EZ) were cultured in complete McCoy’s5A media. The cells were grown in a humidified incubator at 37 °C with 5% CO2 atmosphere.

Tumas S., Meldgaard T.S., Vaaben T.H., Suarez Hernandez S., Rasmussen A.T., Vazquez-Uribe R., Hadrup S.R, & Sommer M.O. (2023). Engineered E. coli Nissle 1917 for delivery of bioactive IL-2 for cancer immunotherapy. Scientific Reports, 13, 12506.

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Broiler Spleen Lymphocyte Proliferation Assay

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The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was
performed based on a previous study by Lee
et al. (2018). After seeding the broiler spleen lymphocytes in
a 96-well plate at a density of 1 × 10⁶ cells per well, the T cell mitogen,
concanavalin A (Con A) (Thermo Fisher Scientific), was added, and the cells were cultured
for 36 h at 37°C in the presence of 5% CO₂. After incubation, MTT (Thermo
Fisher Scientific) was added and the cells were incubated for another 4 h. Finally, the
absorbance was measured at 540 nm.

Park B.M., Lee J., Park Y.K., Yang Y.C., Jung B.G, & Lee B.J. (2023). Immune-enhancing Effects of Chitosan-fermented Feed Additive on Broiler Chickens and Subsequent Protection Conferred against Experimental Infection with Salmonella Gallinarum. The Journal of Poultry Science, 60(2), 2023016.

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Profiling N-Glycosylated Proteins in H4 Cells

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H4 neuroglioma cells were treated with vehicle or 25uM Diltiazem (Sigma) for 4 days, harvested, and extracted in 0.3% CHAPS lysis buffer (0.3% CHAPS, 40mM HEPES pH 7.4, 120 mM NaCl, 1mM EDTA, 10% v/v glycerol). For pulldown of total N-linked glycosylated proteins, 1500 µg lysate was mixed with 20 µg/ml biotinylated Concanavalin A (CON-A) (Vector Laboratories) and the reaction mixture was incubated overnight at 4°C under gentle rotation. To recover CON-A bound proteins, 25 µl neutrAvidin agarose beads (Thermo Fisher Scientific) were added to the reaction mix and samples were incubated at 4°C for 1 hour. The beads were collected by centrifugation at 2500 x g for 2 min, followed by three washes with lysis buffer. N-glycosylated proteins were eluted by boiling the samples at 95°C for 10 min in 2X Laemmli sample buffer. Samples were analyzed by western blot for calnexin (CANX), GCase, and total N-glycosylated proteins by Coomassie brilliant blue staining. Calnexin activity was indirectly assessed by quantifying CANX levels in CON-A pulled down samples.

Stojkovska I., Wani W.Y., Zunke F., Belur N.R., Pavlenko E.A., Mwenda N., Sharma K., Francelle L, & Mazzulli J.R. (2021). Rescue of α-synuclein aggregation in Parkinson’s patient neurons by synergistic enhancement of ER proteostasis and protein trafficking. Neuron, 110(3), 436-451.e11.

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Splenocyte Stimulation and IFN-γ Assay

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Spleens were collected from infected mice and processed as described above to obtain a single cell suspension. Red blood cells were lysed using commercial buffer (BioLegend) per the manufacturer’s instructions. Cells were resuspended in complete Gibco Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Invitrogen) supplemented with 10% FBS, 10 mM HEPES and 50 μM β-mercaptoethanol, then plated at 2 × 10⁵ cells/well and incubated with or without 3 μg of LdAg. Concanavalin A (Con A, ThermoFisher) was used at 2.5 μg/ml to make positive controls. Supernatants were collected after 72 hours and analyzed using the ELISA Max Deluxe Set Mouse IFN-γ from BioLegend.

Helou D.G., Mauras A., Fasquelle F., Lanza J.S., Loiseau P.M., Betbeder D, & Cojean S. (2021). Intranasal vaccine from whole Leishmania donovani antigens provides protection and induces specific immune response against visceral leishmaniasis. PLoS Neglected Tropical Diseases, 15(8), e0009627.

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Quantitative Analysis of Biofilm Biomass

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Biofilms of PA47 were grown on coverslips in the presence or absence of 2% or 4% glucose as described above. Three fluorescent dyes (SYTO9 for total biomass, propidium iodide for dead cells, and ConA for α‐polysaccharides; Thermo Fisher Scientific) were added to each coverslip, and after staining in the dark, samples were imaged using a CLSM (Zeiss LSM 800). The excitation and emission wavelengths for the three dyes were as follows: SYTO9: 633 nm and 650–700 nm; propidium iodide: 458 nm and 460–500 nm; and ConA: 543 nm and 550–600 nm; ImageJ software was used to quantify the biofilm biomass (Qu et al., 2016).

She P., Wang Y., Liu Y., Tan F., Chen L., Luo Z, & Wu Y. (2019). Effects of exogenous glucose on Pseudomonas aeruginosa biofilm formation and antibiotic resistance. MicrobiologyOpen, 8(12), e933.

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Euglena Glycoprotein Purification Protocol

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The resuspended Euglena cells from the culturing (1 ml) were diluted with 5x Binding/Wash buffer (0.25 ml) containing phenylmethylsulfonyl fluoride (2 mM) and lysed by sonication (3 x 10 s, 25% amplitude, 30 s off between each pulse) and centrifuged (5 min, 1000 xg). Not all cells were lysed. Total lysate containing the equivalent of 1.1 mg of protein (Easy Bradford BioRad, BSA standards) was then used for glycoprotein purification using both ConA and WGA Glycoprotein Isolation Kits (Thermo Scientific) according to the manufacturer's instructions. Protein quality was assed using silver stained SDS-PAGE (Bolt 4-12% Bis-TRIS plus, Invitrogen) using SeeBlue Plus2 Pre-stained Protein Standard (Thermo Fisher Scientific) as the standard.

O’Neill E.C. (2021). Glycosylated proteins identified for the first time in the alga Euglena gracilis.

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Membrane Tension Modulation in PtK1 Cells

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PtK1 cells were incubated with either 400 μM dexoycholate (Sigma-Aldrich) for 30 minutes (decreasing membrane tension) or 1 μg/ml concanavalin A (Invitrogen) for 10 minutes (increasing membrane tension) and washed by the media before imaging. HaloTag labeling with TMR ligands was done before membrane tension perturbation.

Lee K., Elliott H.L., Oak Y., Zee C.T., Groisman A., Tytell J.D, & Danuser G. (2015). Functional hierarchy of redundant actin assembly factors revealed by fine-grained registration of intrinsic image fluctuations. Cell systems, 1(1), 37-50.

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