Human PBMCs were isolated from heparinized blood using Histopaque-1077 density gradient (Sigma Aldrich, USA) following a previous protocol with slight modification16 (link). Briefly, fresh blood was diluted with equal volume of PBS (1X) and lightly poured over equal volume of Histopaque 1077 in 15 ml falcon tube followed by centrifugation at 1500 rpm for 15 min at 20 °C in a hanging rotor centrifuge. The middle buffy-coat containing mononuclear cells was collected in a fresh tube and washed twice with PBS. For toxicity assay, 1 × 105 PBMCs/well were seeded into nutrient media in a 96-well culture plate. After 2 h, different concentrations of ADPE were added in each well in triplicate except the control wells and incubated for 24 h. Absorbance and percentage of cytotoxicity were determined as mentioned above using ELISA plate reader (BIORAD-PW41, USA).
Histopaque 1077 density
Manufactured by Merck Group
Sourced in United States
Histopaque-1077 is a sterile, endotoxin-tested solution composed of a polysucrose and sodium diatrizoate. It is designed for the isolation of mononuclear cells from human blood.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Biorad pw41, by Bio-Rad (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Euroclone (1 mentions)
Edta tube, by BD (1 mentions)
Complete rpmi 1640 medium, by Lonza (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Penicillin, by Lonza (1 mentions)
Streptomycin, by Lonza (1 mentions)
Lymphocyte separation medium, by Lonza (1 mentions)
4 protocols using histopaque 1077 density
1
Isolation and Cytotoxicity Assay of PBMCs
2
Culturing Jurkat and Lymphoblast Cells
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Jurkat cell line has been purchased from Sigma-Aldrich. The patient's Lymphoblasts were obtained
from peripheral blood mononuclear cells (PBMCs) isolated by centrifugation over Histopaque 1077 density gradient (Sigma). Jurkat cell lines and patient's Lymphoblasts were cultured in RPMI medium with 10% fetal bovine serum (FBS), supplemented with 100 U/ml penicillin (Gibco), 100 U/ml streptomycin (Gibco) and 2 mM L-glutamine (Euroclone). Cells were cultured at 37° C in a humidified atmosphere with 5% CO2.
from peripheral blood mononuclear cells (PBMCs) isolated by centrifugation over Histopaque 1077 density gradient (Sigma). Jurkat cell lines and patient's Lymphoblasts were cultured in RPMI medium with 10% fetal bovine serum (FBS), supplemented with 100 U/ml penicillin (Gibco), 100 U/ml streptomycin (Gibco) and 2 mM L-glutamine (Euroclone). Cells were cultured at 37° C in a humidified atmosphere with 5% CO2.
3
Isolation and Culture of Splenocytes and PBMCs
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After 30 days of the second boost-up immunization, each group of mice was euthanized and dissected. The spleen was homogenized separately to isolate splenocytes. Then peripheral blood mononuclear cells (PBMCs) were isolated by using the Histopaque-1077 density gradient method (Sigma-Aldrich, Darmstadt, Germany). In brief, the isolated splenocyte of each mouse was diluted at a 1:1 ratio with sterile PBS separately, poured on a 3 mL histopaque-1077 containing tube, and centrifuged at 400× g for 30 min. The separated mononuclear layer was washed twice with sterile PBS (5 mL/wash), counted, and utilized within an hour for further experiments. PBMCs from isolated splenocytes (1 × 106 cells/well) were cultured in six-well plates with the addition of RPMI 1640 medium containing 100 μg/mL penicillin, 100 μg/mL streptomycin, and heat inactivated 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, MA, USA).
4
Isolation of Peripheral Blood Mononuclear Cells
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Mononuclear cells were extracted as previously described [9 (link),19 (link)]. Briefly, less than 10 ml of fresh peripheral blood samples from autistic subjects and control donors were drawn and collected in sterile EDTA tubes (Becton Dickinson, Franklin Lakes, NJ, USA). Peripheral blood mononuclear cells (PMBCs) were isolated by centrifugation over Histopaque 1077 density gradient (Sigma Chemical, St Louis, MO, USA). Briefly, blood was diluted 1:1 in PBS (Sigma, St. Louis, MO, USA), overlaid onto lymphocyte separation media (Lymphocyte Separation Medium -Lonza, Walkersville, MD, USA), centrifuged at 2,200 rpm for 30 minutes at room temperature and plasma was removed. Mononuclear cell fraction was harvested and washed twice in PBS. The final pellet was re-suspended in RPMI 1640 complete medium (Lonza, Verviers, Belgium) containing 10% FBS (EuroClone-Celbio, Milan, Italy), 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin (all Lonza, Verviers, Belgium) and incubated at 37°C with 5% CO2. Lymphocytes (non-adherent cells) were removed.
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