ADCC assay was performed using a modified version of an assay reported previously18 (link),19 (link),45 (link). PBMCs were isolated from human blood (healthy donor) using Ficoll density gradient medium (GE Healthcare) and seeded at 0.5 million/ml in RPMI (10% FBS, 1% Pen/Strep) in 6-well plates. TLR agonists were added to the cells at 1 μM and incubated overnight. Target cells (A549) were pre-labeled with 8 µM CFSE (Biolegend, San Diego, CA) and plated in 96-well plates on the day of PBMC isolation. The next day, cetuximab was added to the wells at 200 nM for 1 h and washed gently. Next, treated PBMCs were counted and added to target cells at the specified ratios and incubated overnight at 37 °C. All samples were set up in triplicates. The supernatant was then analyzed using Pierce LDH Cytotoxicity Assay (Thermofisher, Waltham, MA), and the tumor-cell associated fluorescence intensity was determined at excitation and emission wavelengths of 492 nm and 517 nm, respectively, using Spectramax i3x (Molecular Devices, San Jose, CA).
Ficoll density gradient medium
Manufactured by GE Healthcare
Sourced in Sweden, Australia
Ficoll density gradient medium is a sterile, non-toxic solution used in laboratory settings for the separation and isolation of cells, organelles, and other biological particles based on their density. It is designed to create a density gradient that allows different components to be separated during centrifugation.
Automatically generated - may contain errors
Lab products found in correlation
Carboxyfluorescein succinimidyl ester (cfse), by BioLegend (1 mentions)
Spectramax i3x, by Molecular Devices (1 mentions)
Pierce ldh cytotoxicity assay, by Thermo Fisher Scientific (1 mentions)
Cd4 okt4, by BioLegend (1 mentions)
Cd8 sk1, by BD (1 mentions)
Cd45 hi30, by BioLegend (1 mentions)
Automacs pro separator, by Miltenyi Biotec (1 mentions)
Aim 5 media, by Thermo Fisher Scientific (1 mentions)
4 protocols using ficoll density gradient medium
1
ADCC Assay with PBMC and Cetuximab
2
Isolation and Purification of T-Cell Subsets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Patients’ blood was collected in heparin tubes (40–50 ml in total), and peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Ficoll density gradient medium (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). CD4+ cells and CD8+ cells were isolated from the PBMCs via positive selection using CD4 or CD8 MicroBeads on an autoMACS® Pro Separator (Miltenyi Biotec Norden AB, Lund, Sweden). Flow cytometry was used to determine the purity of some of the sorted T-cell samples, and over 90% of CD45+ cells expressed CD4 (n = 5) or CD8 (n = 5). The following antibodies were used: CD45 (HI30; BioLegend, San Diego, CA, USA), CD4 (OKT4; BioLegend), and CD8 (SK1; BD Biosciences, Stockholm, Sweden).
3
Isolation and Cryopreservation of PBMCs
4
Cytokine Release and Immune Cell Activation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PBMCs were purified from human blood using Ficoll density gradient medium (GE Healthcare) and seeded at 1.5 million cells per well in a 6-well plate. TLR agonists were added to the wells at 1 μM and incubated overnight at 37 °C. The next day, cells were collected and centrifuged at 1200 RPM for 5 min. All samples were set up as duplicates. The supernatant was collected and frozen at − 80 °C until analyzed using the Luminex Human XL Cytokine Discovery Panel (R&D Systems). The cells were used for analysis of NK cell degranulation and T cell activation. Cytokine analysis was performed by the Cytokine Reference Laboratory (University of Minnesota).
For flow cytometry studies, PBMCs were thawed and seeded at 1 million cells/mL per well in a 6 well-plate. 522 and 558 were added to the wells at 1 μM and incubated for 6 h or 24 h at 37 °C. Cells were incubated for 6 h with Brefeldin A for analysis of intracellular cytokines. After incubation, non-adherent cells were collected and stained for T cells (CD3+), NK cells (CD56+) or DCs (CD11c+/CD19−). Once stained, the cells were fixed and permeabilized and the fluorophore conjugated monoclonal antibodies for the intracellular cytokines were added. The cells were stained according to the manufacturer's recommendations and were analyzed by flow cytometry.
For flow cytometry studies, PBMCs were thawed and seeded at 1 million cells/mL per well in a 6 well-plate. 522 and 558 were added to the wells at 1 μM and incubated for 6 h or 24 h at 37 °C. Cells were incubated for 6 h with Brefeldin A for analysis of intracellular cytokines. After incubation, non-adherent cells were collected and stained for T cells (CD3+), NK cells (CD56+) or DCs (CD11c+/CD19−). Once stained, the cells were fixed and permeabilized and the fluorophore conjugated monoclonal antibodies for the intracellular cytokines were added. The cells were stained according to the manufacturer's recommendations and were analyzed by flow cytometry.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!