EDTA-contained blood was used to separate peripheral blood mononuclear cells (PBMC) over a Ficoll-Hypaque cushion (GE, Pittsburgh, PA). Antibodies were incubated with PBMCs at 4°C for 30 min for surface staining. Antibodies were incubated with PBMCs at 4°C for 30 min for surface staining and 30 min for intracellular staining after membrane permeabilization (Fixation/Permeabilization Solution Kit, BD Pharmingen, San Jose, CA). The following fluorochrome-labeled monoclonal antibodies were used: anti-human CD3-percp, anti-CD4-parcific blue, anti-CD8-APC, annexin V-FITC, and isotype control antibodies. Cells were harvested by BD FACSVerse Flow Cytometer (BD Biosciences) and data were analyzed by FlowJo software (Version 10.0.8).
Ficoll hypaque cushion
Manufactured by GE Healthcare
Ficoll-Hypaque cushion is a density gradient medium used for the separation and purification of cells and other biological particles. It is a sterile, endotoxin-tested solution composed of Ficoll and sodium diatrizoate. The product is designed to facilitate the isolation of specific cell populations, such as mononuclear cells, from complex biological samples.
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6 protocols using ficoll hypaque cushion
1
PBMC Isolation and Flow Cytometry Analysis
2
Plasma Processing and PBMC Isolation
3
Comprehensive PBMC Phenotyping by Flow Cytometry
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Peripheral blood mononuclear cells (PBMC) were isolated over a Ficoll-Hypaque cushion (GE, Pittsburgh, PA) from EDTA-contained blood, aliquot, and stored at -80°C before use. Antibodies were incubated with PBMC at 4°C for 30 min for surface staining. After surface staining, the cells were washed and analyzed by flow cytometry. The following fluorochrome-labeled monoclonal antibodies were used (clone): anti-human CD3-percp (OKT3), anti-CD56-APC (B-159), anti-CD16-PEcy7 (3G8), anti-human CD4-BV421 (RPA-T4), anti-human CD107a-BV500 (H4A3), anti-human CD38-FITC (HIT2), anti-human HLA-DR-PE (G46-6), anti-human NKG2D-BV500 (1D11), and Ghost Red 780 (Tonbo Biosciences, San Diego, CA). Mouse IgG1-BV500, IgG1-APC and IgG2a-PE isotype antibodies were used to gate on CD107a/NKG2D-BV500, CD38 and HLA-DR respectively. Cells were collected by BD FACSVerse Flow Cytometer (BD Biosciences) and data were analyzed by FlowJo software (Version 10.0.8).
4
Cytokine response of PBMCs to oral bacteria
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Peripheral blood mononuclear cells (PBMCs) from healthy individuals were isolated over a Ficoll-Hypaque cushion (GE, Pittsburgh, PA). After the bacteria were heat-inactivated at 60°C for 30 min, heat-inactivated A. meyeri, A. odontolyticus, and N. elongata were added to cell culture at a final concentration of 1 × 107 CFU/ml; LPS from E.coli 055:B5 (LPS-B5, invovoGen) was used as a positive control with a final concentration of 2 ng/ml. PBMCs were cultured with brefeldin A (5 μg/ml, BD) and incubated at 37°C for 5 h. Cells were then collected and washed with PBS, followed by a 20 min incubation with 50 μl aqua blue (Life Technologies, Carlsbad, CA) at 4°C to exclude dead cells. Next, 50 μl of an antibody cocktail containing anti-human CD3 (RRID:AB_2869824) and anti-human CD14 (RRID:AB_396848) were used for surface staining. After washing and permeabilization, cells were intracellularly stained with anti-human TNF-α (RRID:AB_2204110), anti-human IL-1β (RRID:AB_400438), and anti-human IL-6 (RRID:AB_397228). Fluorescence-labeled antibodies were purchased from BD or Biolegend (San Diego, CA). After washing, cells were collected and analyzed using a BD FACSVerse flow cytometer (BD). Data were analyzed using the FlowJo software (Version 10.0.8).
5
Isolating Primary Human T Cells
6
PBMC Cytokine Response to Bacterial Stimuli
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Peripheral blood mononuclear cells (PBMCs) from healthy individuals were isolated over a Ficoll-Hypaque cushion (GE, Pittsburgh, PA). After the bacteria were heat-inactivated at 60°C for 30 min, Massilia timonae, Haemophilus parainfluenzae, and Anaerococcus prevotii were added to cell culture at a final concentration of 5 × 106/ml; LPS from Escherichia coli 055:B5 was used as a positive control with a final concentration of 2 μg/ml. PBMCs were cultured with brefeldin A (5 μg/ml, BD) and incubated at 37°C for 6 h. Cells were then collected and washed with PBS, followed by a 20-min incubation with 50 μl aqua blue (Life Technologies, Carlsbad, CA, United States) at 4°C to exclude dead cells. Next, 50 μl of an antibody cocktail containing anti-human CD3 (OKT3), anti-human CD14 (M5E2), and anti-human CD16 (3G8) were used for surface staining. After washing and permeabilization, cells were intracellularly stained with anti-human TNF-α (MAb11), anti-human IL-1β (AS10), and anti-human IL-6 (MQ2-6A3). Fluorescence-labeled antibodies were purchased from BD or Biolegend (San Diego, CA). After washing, cells were collected and analyzed using a BD FACSVerse flow cytometer (BD). Data were analyzed using the FlowJo software (version 10.0.8).
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