Freshly collected PBMCs from the five donors were plated in 48-well plates (1 million cells/well) containing T cell media: RPMI-1640 medium (Gibco) with 5% human serum (Sigma-Aldrich), 1% Pen-Strep (Thermo Fisher Scientific), 1X MEM non-essential amino acids solution (Sigma-Aldrich), 1X sodium pyruvate (Gibco), and 1X HEPES buffer (Sigma-Aldrich), in addition to 1.6μL of Phytohemagglutinin-L (PHA-L) per well (500X, Invitrogen; Cat. no.: 00-4977-93) and 20 ng/mL of IL2 (BioLegend), and placed in a 5% CO2 incubator at 37 °C. Cells were split every 2–3 days depending on density, and IL2 was replenished at a concentration of 20 ng/mL on the first two splits, then downscaled to 10 ng/mL. Cells were expanded for 12 days and were validated by flow cytometry using CD3-PE, CD4-PE and CD8-PB antibodies (Supplementary Fig. S6 ).
Phytohemagglutinin l pha l
Manufactured by Thermo Fisher Scientific
Phytohemagglutinin-L (PHA-L) is a lectin protein derived from the red kidney bean (Phaseolus vulgaris). It functions as a potent mitogen, capable of stimulating the proliferation of T lymphocytes in cell culture.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Hepes buffer, by Merck Group (1 mentions)
Human serum, by Merck Group (1 mentions)
Interleukin 2 (il 2), by BioLegend (1 mentions)
Mem non essential amino acids solution, by Merck Group (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 2mm glutamine, by Thermo Fisher Scientific (1 mentions)
2 protocols using phytohemagglutinin l pha l
1
Expansion of Human T Cells from PBMCs
2
Culturing Jurkat Cells and Isolating PBMCs
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The human leukemia cell line Jurkat was maintained in RPMI 1640 2 mM glutamine medium (Gibco) supplemented 10% heat-inactivated fetal bovine serum (Invitrogen), 100 U/mL penicillin and 100 µg/mL streptomycin (Invitrogen). Cells were cultured in a humidified incubator at 37°C and 5% CO2.
Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats from healthy donors (Sanquin) by density gradient centrifugation using Lymphoprep (Nycomed Pharma). Recovered cells were washed twice in sterile PBS with 0.1% bovine serum albumin and resuspended in RPMI-1640 2 mM glutamine (Gibco), containing 10% heat-inactivated fetal bovine serum (Invitrogen), 100 U/mL penicillin and 100 µg/mL streptomycin (Invitrogen). When indicated, PBMCs were treated with 1 µg/mL phytohemagglutinin-L (PHA-L, Invitrogen) for 24 hours prior to stimulation with galectins/cytokines. All studies involving human samples were conducted following the Declaration of Helsinki principles and current legislation on the confidentiality of personal data.
Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats from healthy donors (Sanquin) by density gradient centrifugation using Lymphoprep (Nycomed Pharma). Recovered cells were washed twice in sterile PBS with 0.1% bovine serum albumin and resuspended in RPMI-1640 2 mM glutamine (Gibco), containing 10% heat-inactivated fetal bovine serum (Invitrogen), 100 U/mL penicillin and 100 µg/mL streptomycin (Invitrogen). When indicated, PBMCs were treated with 1 µg/mL phytohemagglutinin-L (PHA-L, Invitrogen) for 24 hours prior to stimulation with galectins/cytokines. All studies involving human samples were conducted following the Declaration of Helsinki principles and current legislation on the confidentiality of personal data.
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