ELISPOT plates (Millipore, Bedford, MA) were precoated with 5µg/mL of capture antibodies against gamma-IFN or IL-17 (Mabtech, Nacka Strand, Sweden) in phosphate-buffered saline (PBS) and stored overnight at 4°C. The responding cells separated from fresh PBMCs were co-cultured with an equal number of irradiated donor or autologous PBMCs as stimulating cells (1.5 × 105 cells/well), or unstimulated in medium alone, or with phytohemaglutinin at 1 µg/mL (Sigma–Aldrich Corp, St. Louis, MO). After 44 h incubation at 37°C, the plates were washed and biotinylated detection antibodies (Mabtech) were added for 2 h at room temperature. After five washes with PBS, streptavidin–horseradish-peroxidase conjugate in PBS BSA 0.5% (Dako, Glostrup, Denmark) was added for 1 h at room temperature, followed by five washes. Finally, 100 µL/well of 3,3,5,5-tetramethylbenzidine liquid substrate (Sigma–Aldrich) was added and incubated for 15 min in the dark. The resulting spots were counted with an ELISPOT image analyzer (CTL, Inc., Cleveland, OH), as previously described (7 ,19 (link),20 (link)).
Phytohemaglutinin
Manufactured by Merck Group
Sourced in United States
Phytohemaglutinin is a lectin derived from the red kidney bean (Phaseolus vulgaris). It is used in various laboratory applications as a cell culture supplement to stimulate the proliferation of T lymphocytes.
Automatically generated - may contain errors
Lab products found in correlation
Elispot plate, by Merck Group (1 mentions)
Biotinylated detection antibody, by Mabtech (1 mentions)
3 3 5 5 tetramethylbenzidine liquid substrate, by Merck Group (1 mentions)
Streptavidin horseradish peroxidase conjugate in pbs bsa 0.5, by Agilent Technologies (1 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (1 mentions)
Concanavalin a (cona), by Merck Group (1 mentions)
Antibiotic antimycotic solution, by HiMedia (1 mentions)
L glutamine, by HiMedia (1 mentions)
4 protocols using phytohemaglutinin
1
Quantifying Cytokine-Producing Cells Using ELISPOT
2
Mesenchymal Stem Cell Immunomodulation
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MSCs were cultured in proliferative medium containing 5% human serum (SAB, SAB400, SAB1000, SABH2O2, or PS) for 3 days. They were then trypsinized and plated with 2 × 105 PBMC at different densities to get ratios of 1/5, 1/10, and 1/50 (MSC/PBMC) in 96-well plates. Cells were cultured in IMDM (Invitrogen) containing 10% heat inactivated FCS, 100 µg/mL penicillin/streptomycin, 2 mM glutamine, 0.1 mM non-essential amino acids, 5 × 105 M 2-mercaptoethanol, 1 mM sodium pyruvate, 10% FCS, 25 mM HEPES, and 2.5 µg/mL phytohemaglutinin (Sigma) for T lymphocyte activation. After 3 days, T lymphocyte proliferation was measured with Cell Proliferation ELISA, BrdU assay (Sigma-Aldrich). Results were expressed as the percentage of proliferation ± SEM and normalized at 100% for proliferation of activated PBMC minus basal proliferation.
3
Lymphocyte Activation and Proliferation
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Phorbol myristate acetate (PMA), tetrazolium dyes, NBT (Nitroblue Tetrazolium) and MTT [3-(4, 5-dimethylthiozol-2-yl)-2, 5 diphenyl tetrazolium bromide], mitogens [concanavalin A (Con A), phytohemaglutinin (PHA) and lipopolysaccharide (LPS)] and Culture medium (RPMI-1640) were procured from Sigma, USA. Antibiotic-Antimycotic solution, L-glutamine, fetal bovine serum (FBS), lymphocyte separation medium (Hisep), dimethyl sulfoxide (DMSO) were purchased from Himedia Laboratories Pvt. Ltd. (India). The culture medium was supplemented with 5% heat inactivated FBS, Antibiotic-Antimycotic solution, 10mM HEPES and 50μM Mercaptoethanol. All other chemicals and solvents used were analytical grade.
4
Evaluating Lymphocyte Proliferation by Flow Cytometry
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The specific proliferation
of different lymphocyte subpopulations
was evaluated using, as APCs, autologous moDCs pre-stimulated with
10 nM of different experimental combinations for 48 h previously described.
Proliferation was determined using a 5,6-carboxyfluorescein diacetate N-succinimidyl ester (CFSE) dilution assay (Thermo Fisher
Scientific). A total of 1.5 × 106/mL pre-labeled CD14– cells were cultured with moDCs pre-stimulated
in different experimental combinations (10:1 ratio) at a final volume
of 250 μL of complete medium in 96-well plates for 6 days at
37 °C and 5% CO2. 10 μg/mL phytohemaglutinin
(Sigma-Aldrich) was used as positive proliferative control and unstimulated
moDCs as negative proliferative control. The proliferative responses
were assessed by flow cytometry, analyzing CFSElow expression in the
different cell subsets as T-lymphocytes (CD3+T, CD4+T and CD8+T), B-lymphocytes (CD19+B)
and NK-cells (CD56+NK). Results were expressed as PI. The
PI was calculated for each cell subset as ([% CFSElow stimulated PBMCs
+ DC] – [% CFSElow stimulated PBMCs)/(% CFSElow unstimulated
PBMCs + DCs)].69 (link),70 (link) In addition, the percentages
of regulatory T (Treg) cells were also evaluated by flow cytometry,
analyzing the expression of the CD4+CD127–/lowCD25+FOXP3+ markers.
of different lymphocyte subpopulations
was evaluated using, as APCs, autologous moDCs pre-stimulated with
10 nM of different experimental combinations for 48 h previously described.
Proliferation was determined using a 5,6-carboxyfluorescein diacetate N-succinimidyl ester (CFSE) dilution assay (Thermo Fisher
Scientific). A total of 1.5 × 106/mL pre-labeled CD14– cells were cultured with moDCs pre-stimulated
in different experimental combinations (10:1 ratio) at a final volume
of 250 μL of complete medium in 96-well plates for 6 days at
37 °C and 5% CO2. 10 μg/mL phytohemaglutinin
(Sigma-Aldrich) was used as positive proliferative control and unstimulated
moDCs as negative proliferative control. The proliferative responses
were assessed by flow cytometry, analyzing CFSElow expression in the
different cell subsets as T-lymphocytes (CD3+T, CD4+T and CD8+T), B-lymphocytes (CD19+B)
and NK-cells (CD56+NK). Results were expressed as PI. The
PI was calculated for each cell subset as ([% CFSElow stimulated PBMCs
+ DC] – [% CFSElow stimulated PBMCs)/(% CFSElow unstimulated
PBMCs + DCs)].69 (link),70 (link) In addition, the percentages
of regulatory T (Treg) cells were also evaluated by flow cytometry,
analyzing the expression of the CD4+CD127–/lowCD25+FOXP3+ markers.
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