Dual-color fluorospot kits for IFNγ and IL2 (Mabtech) were used as per manufacturer’s instructions. PBMC were thawed and rested over night. Cells with viability ≥70% before and after resting were added at 250,000 PBMC/well in 100 μL of RPMI 1640 with glutamine (Gibco) containing 10% human AB serum (Nabi) and 1% antibiotics (Gibco) and stimulated in duplicate wells with HIV inactivated virions and control (gift of Dr. Jeff Lifson [45 (link)]; 6 μg/mL), VZV inactivated cell lysate and mock-infected control prepared as previously described [46 (link)] at a pre-optimized concentration, phytohemagglutinin A (PHA; Sigma; 0.01 μg/mL) or anti-CD3 and anti-CD28 mAb (CD3/CD28; Mabtech; 0.1 μg/mL). After 36 h at 37°C in a 5% CO2 humidified atmosphere, plates were washed; bound IFNγ was detected with 7-B6-1-FS FITC and bound IL-2 with 11-Biotin. Spots were revealed using a mixture of anti-FITC-Green fluorochrome (IFNγ) and SA-Red fluorochrome (IL-2) and analyzed with an Immunospot II plate reader (CTL). Results were reported as mean spot-forming cells (SFC)/105 PBMC in antigen- or mitogen-stimulated wells after subtraction of the mean SFC in control wells.
Rpmi 1640 with glutamine
Manufactured by Thermo Fisher Scientific
Sourced in Canada, United States
RPMI 1640 with glutamine is a widely used cell culture medium formulation. It provides essential nutrients and supports the growth of a variety of cell types. The medium includes glutamine, an important amino acid required for cell proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Phytohemagglutinin (pha), by Merck Group (1 mentions)
Co2 incubator, by Thermo Fisher Scientific (1 mentions)
Magpix multiplex reader, by Bio-Rad (1 mentions)
Vacutainer tube, by BD (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Mem non essential amino acid, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
6 protocols using rpmi 1640 with glutamine
1
Dual-Color Fluorospot Assay for IFNγ and IL-2
2
Isolation and Culture of Human Bone Marrow Mesenchymal Stem Cells
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The RPMI-8226 human multiple myeloma cell lines (gift from Professor Chen Zi-xing, Hematology Institute, Suzhou) were cultured with RPMI-1640 with glutamine (GIBCO) supplemented with 10% (v/v) fetal bovine serum (GIBCO) and 1% (w/v) penicillin/streptomycin in a humidified 5% CO2 incubator (Thermo) at 37°C. RPMI-8226 cells were counted by automatic cell counting system to assay the proliferation.
1 mL of bone marrow sample was collected from young adult donor using a needle, which was immediately transferred into a sterile tube containing 1% heparin solution. To isolate bone marrow mononuclear cells, the samples were centrifuged in a 1.077 g/mL Ficoll density gradient for 30 min. The cells in the white middle layer were isolated and resuspended in culture medium with DMEM (GIBCO) containing 10% (v/v) fetal bovine serum (GIBCO) and 1% (w/v) penicillin/streptomycin. Then 1 × 106 cells were seeded in a 100 mm dish that was precoated with 0.1% gelatin and incubated at 37°C with 5% CO2 at 100% humidity. After 3 days, the medium containing floating cells was removed and new medium was added to the remaining adherent cells. These adherent cells were considered to be BMSCs. The medium was changed every 3 days. The third passages were used for the following experiments [5 (link), 6 (link)].
1 mL of bone marrow sample was collected from young adult donor using a needle, which was immediately transferred into a sterile tube containing 1% heparin solution. To isolate bone marrow mononuclear cells, the samples were centrifuged in a 1.077 g/mL Ficoll density gradient for 30 min. The cells in the white middle layer were isolated and resuspended in culture medium with DMEM (GIBCO) containing 10% (v/v) fetal bovine serum (GIBCO) and 1% (w/v) penicillin/streptomycin. Then 1 × 106 cells were seeded in a 100 mm dish that was precoated with 0.1% gelatin and incubated at 37°C with 5% CO2 at 100% humidity. After 3 days, the medium containing floating cells was removed and new medium was added to the remaining adherent cells. These adherent cells were considered to be BMSCs. The medium was changed every 3 days. The third passages were used for the following experiments [5 (link), 6 (link)].
3
Whole Blood Stimulation Assay
4
Plasma and Blood Cell Preparation
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Venous blood samples were drawn from subjects into sodium citrate-buffered Vacutainer tubes (BD Diagnostic, Spain) and coated tubes. Plasma was prepared at room temperature by centrifugation (15 min at 1500× g). The supernatant was stored in 1.5-mL tubes at −80 °C until use. By contrast, whole blood cells (erythrocytes and total leukocytes) were obtained following a previously described procedure [46 (link)]. For this, 500 µL of the peripheral blood were diluted with 500 µL of RPMI 1640 medium without glutamine (Gibco, Burlington, ON, Canada) and 10 µL gentamicin (0.1 mg/mL in the tube). The samples were incubated for 4 h at 37 °C in a saturated atmosphere of CO2 and humidity. Afterward, samples were centrifuged at 900× g for 10 min to obtain the whole blood cell pellets after plasma removal. RPMI 1640 with glutamine (Gibco, Canada) was added to the blood cells to make 1 mL. These aliquots were stored at –80 °C until they were used for determining the MDA levels.
5
Epidermal Growth Factor Receptor Signaling
6
Deciphering CLL Molecular Subtypes
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In the present study, a total of 70 CLL patients (35 IGHV-unmutated samples + 35 IGHV-mutated samples) were included. All patients were diagnosed according to recently revised criteria [25 (link)] and the tumor samples were collected at the time of diagnosis. The patients in the study were included from different hematology departments in the western part of Sweden after written consent had been obtained. Only CLL peripheral blood mononuclear cells (PBMC) samples with a tumor percentage of leukemic cells ≥70 % were selected in this study. Clinical and molecular data are summarized in Additional file 1 A and B. PBMCs from peripheral blood of age-matched normal healthy controls was prepared using the Ficoll extraction method and normal CD+19 positive sorted B cell DNA from eight healthy age-matched controls were bought from a company (3H Biomedicum, Uppsala, Sweden). Two CLL cell lines (HG3 [26 (link)] and MEC1 [27 (link)]) and one Burkitt lymphoma B cell line (RAMOS) [28 (link)] were used for DAC treatment experiments. All cell lines were cultured in RPMI 1640 with glutamine (Invitrogen, Carlsbad, USA) supplemented with 10 % fetal bovine serum and 1× penicillin/streptomycin (FBS; Invitrogen, Carlsbad, USA).
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