Mouse spleens were homogenized by grinding the spleens between the frosted ends of two sterile microscope slides. The resulting homogenate was suspended in 10 mL of complete R10 culture medium (RPMI-1640 containing 10% fetal bovine serum [FBS] and 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin sulfate, 1 mM sodium pyruvate, 1 mM HEPES, 100 μM non-essential amino acids) and ground into single cells. The splenocytes were subsequently isolated by Ficoll-Hypaque density gradient centrifugation and resuspended for 24 hr in complete R10 culture medium containing either 1 mg/mL concavalin-A (Con-A; Sigma), 2 mg/mL of HPV16 E7 and E6, as well as VSV N peptide pools, consisting of 15-mer peptides with 11-mer overlap, covering the entire protein sequence of HPV16 E7, E6, and VSV N. Splenocytes cultured with medium alone were used as control. Input cell numbers were 4 × 105. Input splenocytes per well were assayed in duplicate wells using a mouse IFNγ ELISPOT kit (BD Biosciences, San Diego, CA). The resulting spots were counted using an Immunospot Reader (CTL, Cleveland, OH). Peptide pool-specific IFNγ [no hyphen, also with beta] responses were considered positive if the response (minus media background) was ≥3 fold above the media response and ≥50 SFC/106 splenocytes.
Concavalin a con a
Manufactured by Merck Group
Sourced in United States
Concanavalin-A (Con-A) is a lectin protein that is derived from the jack bean (Canavalia ensiformis). It has the ability to agglutinate (clump together) certain types of cells, including red blood cells and lymphocytes. Con-A is commonly used as a tool in various biological and immunological applications.
Automatically generated - may contain errors
Lab products found in correlation
Lipopolysaccharide lps, by Merck Group (2 mentions)
Mouse ifn γ elispot kit, by BD (1 mentions)
5 and 6 carboxyfluorescein diacetate succinimidyl ester cfse, by Thermo Fisher Scientific (1 mentions)
Mouse cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Cfse cell proliferation kit, by Thermo Fisher Scientific (1 mentions)
Mouse il 12 il 23 total p40 elisa ready set go, by Thermo Fisher Scientific (1 mentions)
Mouse il 2 elisa set, by BD (1 mentions)
Mouse ifn gamma elisa ready set go, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Ril 2, by Thermo Fisher Scientific (1 mentions)
Rotenone, by Merck Group (1 mentions)
Catechin, by Merck Group (1 mentions)
Epicatechin, by Merck Group (1 mentions)
6 protocols using concavalin a con a
1
Splenocyte Isolation and Stimulation Assay
2
T. spiralis-Sensitized CD4+ T Cell Activation by rTsPmy-Pulsed DCs
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To determine whether rTsPmy-pulsed DCs could activate T. spiralis-sensitized CD4+ T cells, the DCs pulsed with rTsPmy for 72 h, and then were co-cultivated with T. spiralis-sensitized CD4+ T cells, the T. spiralis-sensitized CD4+ T cells were obtained from the spleens of BALB/c mice infected with 400 T. spiralis ML for 60 days using magnetic-activated cell sorting (MACS) with a mouse CD4+ T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). A total of 5 × 104 or 2.5 × 104 DCs were plated in each well of round-bottom 96-well plates and then co-cultivated with 5 × 105T. spiralis-sensitized CD4+ T cells stained with 5-and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) (eBioscience, San Diego, CA, USA), in the presence of 5 μg/ml Concavalin-A (Con-A) (Sigma-Aldrich, St. Louis, MO, USA) which is a nonspecific stimulator for mouse T cells. Subsequently, the proliferation of T cells was measured by fluorescence-activated cell sorting (FACS).
To determine the cytokine production, 5 × 104 DCs were plated in each well of round-bottom 96-well plates and co-incubated with 5 × 105T. spiralis-sensitized CD4+ T cells for 36 h, then supernatants were collected and cytokines measured by ELISA as described above.
To determine the cytokine production, 5 × 104 DCs were plated in each well of round-bottom 96-well plates and co-incubated with 5 × 105T. spiralis-sensitized CD4+ T cells for 36 h, then supernatants were collected and cytokines measured by ELISA as described above.
3
Splenocyte Proliferation Analysis
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The proliferative splenocytes were tracked by the intracellular dye carboxyfluorescein diacetate succinimidyl ester (CFSE) (CFSE cell proliferation kit, Invitrogen, Waltham MA, USA, Molecular Probes), diluted in DMSO according to the manufacturer instructions. The assay was performed as previously described [24 (link)]. Splenocytes were separated by the stimulus groups (peptide 2, SMVYLLIGYL) at a dilution of 10−5, mock (culture medium RPMI) or positive control (concavalin A (ConA) (4 μg/mL, Sigma-Aldrich, St. Louis, MO, USA)).
4
Cytokine Profiling of Murine Splenocytes
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The suspension of spleen cells from immunized mice was prepared by mechanical disruption. Ammonium chloride lysing buffer was used for the lysis of red blood cells. The prepared cells were seeded into 96-well cell culture plates at a density of 1 × 106 cells/mL and stimulated either with VLPs (HaVP1-PADRE-4, TSPyV VP1 and HaPyV VP1) at a concentration 5 μg/mL or polyclonal activators Concavalin A (ConA) (Sigma-Aldrich) and lipopolysaccharide (LPS) (Sigma-Aldrich) added at concentrations 3 μg/mL and 1 μg/mL, respectively, to the growth medium: RPMI-1640 containing 10% FBS (Merck-Millipore) and antibiotics. After 24 h and 48 h of incubation, culture supernatants were collected and stored at −80 °C until analysis.
Interleukin 12 (IL-12), interleukin 2 (IL-2) and interferon gamma (IFN-γ) concentrations in mouse spleen cell cultures were determined by sandwich ELISA using Mouse IL-12/IL-23 total p40 ELISA Ready-Set-Go, Mouse IFN gamma ELISA Ready-Set-Go (eBioscience, San Diego, CA, USA) and Mouse IL-2 ELISA set (BD Biosciences, San Jose, CA, USA) according to the recommendations of the suppliers.
Interleukin 12 (IL-12), interleukin 2 (IL-2) and interferon gamma (IFN-γ) concentrations in mouse spleen cell cultures were determined by sandwich ELISA using Mouse IL-12/IL-23 total p40 ELISA Ready-Set-Go, Mouse IFN gamma ELISA Ready-Set-Go (eBioscience, San Diego, CA, USA) and Mouse IL-2 ELISA set (BD Biosciences, San Jose, CA, USA) according to the recommendations of the suppliers.
5
Naive and Memory-like CD4+ T Cell Responses
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Naïve CD4 + T cells were purified from mouse spleens using Negative Isolation Kit (Dynal Biotech). Cell purity was >95% as measured by flow cytometry. For cell culture, we used complete media containing 20 ng/ml human recombinant interleukin-2 (rIL-2, PeproTech). Naïve CD4 + T cells (10 6 /ml) were stimulated with concavalin A (ConA;
1.5 μg/ml, Sigma) or with IL-12 (10 ng/ml) plus IL-18 (10 ng/ml) for indicated time points. For memory-like differentiation, naïve CD4 + T cells were first stimulated with ConA (1.5 μg/ml) for 24h, washed and cultured (0.5 x 10 6 /ml) in presence of 20 ng/ml rIL-2 for 6 days. Differentiated memory-like cells were stimulated with ConA or with IL-12 (10 ng/ml) plus IL-18 (10 ng/ml) for indicated time points. DPI (5 μM; Sigma), rotenone (2.5 μM; Sigma) or antimycin A (4 μM) were added to cell culture 30 min before the stimulus.
1.5 μg/ml, Sigma) or with IL-12 (10 ng/ml) plus IL-18 (10 ng/ml) for indicated time points. For memory-like differentiation, naïve CD4 + T cells were first stimulated with ConA (1.5 μg/ml) for 24h, washed and cultured (0.5 x 10 6 /ml) in presence of 20 ng/ml rIL-2 for 6 days. Differentiated memory-like cells were stimulated with ConA or with IL-12 (10 ng/ml) plus IL-18 (10 ng/ml) for indicated time points. DPI (5 μM; Sigma), rotenone (2.5 μM; Sigma) or antimycin A (4 μM) were added to cell culture 30 min before the stimulus.
6
Hazelnut Skin and Blackcurrant Leaf Analysis
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Skins from hazelnut (Corella avellana; hereafter referred to as HN) were provided by Dr Hervé Hoste (INRA, Toulouse, France). Leaves from blackcurrant (Ribes nigrum; hereafter referred to as BC) were sourced from Goring-upon-Thames, UK. Catechin, epiCatechin, concavalin A (con A) and lipopolysaccharide (LPS) were obtained from Sigma-Aldrich (Schnelldorf, Germany).
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