To assess effects of the different agents on the proliferative capacity of T cells, PBMC (106/mL) were seeded into wells of 96 well cluster plates (100 μl per well). To induce polyclonal T cell proliferation, wells were precoated with mouse anti-human CD3 antibody (1 μg mL–1; clone HIT3a), and mouse anti-human CD28 antibody (1 μg mL–1; clone CD28.2) was added to PBMC prior to seeding (both antibodies from Biolegend, San Diego, CA). In parallel settings, T cells were stimulated using phytohemagglutinin (PHA) isolated from Phaseolus vulgaris (Sigma-Aldrich) at a final concentration of 5 μg mL–1. PBMC left unstimulated served as internal controls. The different agents were applied to triplicates at the concentrations indicated. T cell proliferation was assayed as genomic incorporation of 3H-thymidine (0.5 μCi per well) added on day 3 of culture. On the next day, cells were harvested onto glass fiber filters. Retained radioactivity was detected using a β counter (1205 Betaplate, LKB Wallac, Turcu, Finnland).
Phaseolus vulgaris
Manufactured by Merck Group
Phaseolus vulgaris is a common type of bean used in laboratory equipment. It serves as a model organism for various scientific studies and experiments.
Automatically generated - may contain errors
4 protocols using phaseolus vulgaris
1
T Cell Proliferation Assay
2
Avian Proinflammatory Potential via PHA-P
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To determine birds in-vivo general proinflammatory potentiall42 , the responses to PHA-P, a lectin from Phaseolus vulgaris (Sigma Chemical, St. Louis, MO), was measured in the wing web of each bird following the methods described elsewhere42 –45 . Briefly, on day 1, a 0.05 ml intradermal injection of a solution of 1 mg/ml PHA-P in phosphate saline buffer (PBS) was injected in the wing web of each bird. The dermal swelling response was measured as the percentage increase in wing web thickness at the injection site 24 h post-PHA-P injection (day 2). Measurements were recorded to the nearest 0.01 mm using a mechanic digital micrometre.
3
Lectin Sourcing and Preparation
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Lectins from: Pisum sativum (L5380), Arachis hypogaea (L0881), Triticum vulgaris (L9640), Glycine max (L1395), Phaseolus vulgaris (61764), Agaricus bisprous (L5640), Lycopersicon esculentum (L2886) were purchased from Sigma-Aldrich, dissolved in sterile phosphate-buffered saline (PBS), and stored at 4°C in a concentration of 1 mg/mL. Succinyl-WGA (W0110) and wheat germ agglutinin FITC-conjugate (L4895), were purchased at Vector Laboratories and Sigma-Aldrich, respectively. These variants were also dissolved in sterile phosphate-buffered saline (PBS) and stored at 4°C in a concentration of 1 mg/mL. Lectin from Sambucus nigra (ZB0106) was purchased from Vector Laboratories. Detailed information on each lectin is included in Table 1 and obtained from Sigma-Aldrich product sheets.
4
Evaluating Cell-Mediated Immunity in Birds
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To determine cell-mediated immunity, the responses to PHA-P (a lectin from Phaseolus vulgaris (Sigma Chemical, St. Louis, MO)), was measured in the wing web following the methods described elsewhere (Nazar & Marin, 2011; Roberts et al., 2009) . Briefly, on day 1, a 0.05 ml of a solution of PHA-P in phosphate saline buffer (1 mg/ml) was injected intradermally in the wing web of each bird. The dermal swelling response was measured as the percentage increase in wing web thickness at the injection site 24-h post-PHA-P injection (day 2). Measurements were recorded to the nearest 0.01 mm using a mechanical digital micrometer.
Heterophil/lymphocyte and innate/acquired ratio Leukocyte counts were performed on blood smears stained with the May-Gru ¨nwald-Giemsa method. Differential counts of 100 white cells per blood smear were made (Huff et al., 2005; Fair et al., 1999; Nazar & Marin, 2011) . The INN/ACQ (used to compare the subpopulations of cells involved in the two main branches of the immune response) cell and heterophil/lymphocyte (H/L) ratios (commonly used as a hematological indicator of chronic stress) were calculated using the following formulae: INN/ACQ ¼ (number of basophils + number of heterophils + number of monocytes) / (number of eosinophils + number of lymphocytes); H / L ¼ (number of heterophils) / (number of lymphocytes).
Heterophil/lymphocyte and innate/acquired ratio Leukocyte counts were performed on blood smears stained with the May-Gru ¨nwald-Giemsa method. Differential counts of 100 white cells per blood smear were made (Huff et al., 2005; Fair et al., 1999; Nazar & Marin, 2011) . The INN/ACQ (used to compare the subpopulations of cells involved in the two main branches of the immune response) cell and heterophil/lymphocyte (H/L) ratios (commonly used as a hematological indicator of chronic stress) were calculated using the following formulae: INN/ACQ ¼ (number of basophils + number of heterophils + number of monocytes) / (number of eosinophils + number of lymphocytes); H / L ¼ (number of heterophils) / (number of lymphocytes).
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