Blood samples of 0.25 mL were taken from the left femoral artery to check immune cells and endothelial damage according to syndecan-1 and cytokine levels at T0, T3, and T6. The blood was collected in tubes precoated with ethylenediaminetetraacetic acid (EDTA). Peripheral blood mononuclear cells (PBMCs) were isolated from the blood using density-gradient centrifugation over a Biocoll gradient solution (Biochrom Ltd., Cambridge, UK). After sacrifice, the heart, lungs, and kidneys were extracted and minced into 1 mm3 (link) pieces on ice. After washing, the tissues were digested with 1 mg/mL collagenase type 1 (Sigma-Aldrich, St. Louis, MO, USA) in 5 mL phosphate-buffered saline (PBS) at 37 °C for 60 min. After incubation, mononuclear cells in the digested solution were filtered through a 70 µm cell strainer (SPL Life Science Co., Pocheon, Korea).
Biocoll gradient solution
Manufactured by Harvard Bioscience
Sourced in Germany
Biocoll gradient solution is a laboratory reagent used for the separation and purification of cells and cellular components. It is a density gradient medium that allows the separation of different cell types or subcellular fractions based on their density. The solution is designed to create a continuous density gradient, enabling the effective isolation and enrichment of target samples.
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6 protocols using biocoll gradient solution
1
Comprehensive Immunological and Tissue Analysis
2
Isolation and Characterization of Immune Cells
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After the rats were euthanized, the skull was cut from anterior to posterior along the dorsal midline, and the brain was separated and fixed in 4% paraformaldehyde for immunohistochemical assay. The abdomen was dissected, and the liver and the kidneys were isolated. Blood samples were obtained from an abdominal artery puncture with heparin-coated 2 mL syringes and collected into tubes precoated with ethylenediaminetetraacetic acid (EDTA). The liver and the kidneys tissues were divided into two parts. One was fixed in 4% paraformaldehyde for immunohistochemical assay and the other was minced into 1 mm3 pieces on ice. After the minced tissues were washed, the tissues were digested with 1 mg/mL collagenase type 1 (Sigma-Aldrich, St. Louis, MO, USA) in 5 mL phosphate-buffered saline (PBS) at 37°C for 60 min. After incubation, the cells in the digested solution were filtered through a 70 µm cell strainer (SPL Life Science, Seoul, Korea). Lymphocytes were isolated from the blood, the liver and the kidneys, to assess ER stress and to detect ROS, using density-gradient centrifugation over a Biocoll gradient solution (Biochrom, Berlin, Germany).
3
Quantifying Intracellular Oxidative Stress
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The blood in the EDTA tube was transferred to a conical tube and diluted to 10 mL with phosphate-buffered saline (PBS; Gibco, USA). Peripheral blood mononuclear cells (PBMCs) were isolated from the blood using density-gradient centrifugation over a Biocoll gradient solution (Biochrom, German). The PBMCs were washed with fluorescence activated cell sorter (FACS) buffer [1% bovine serum albumin (BSA) and 0.01% NaN3 in PBS]. After washing the PBMCs, they were stained with 2',7'-dichlorofluorescein diacetate (H2DCFDA, Life Technologies, USA), changing from non-fluorescent into fluorescent on oxidation, to detect intracellular ROS. The staining was performed for 30 minutes in the dark at room temperature and the sample was analysed on a flow cytometer. The data were analysed using FlowJo software (Tree Star, USA).
4
PBMC Isolation from Cancer Patients
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Heparin-anticoagulated peripheral blood samples were obtained from each patient with cancer, and PBMCs were isolated by density gradient centrifugation using Biocoll gradient solution (Biochrom, Berlin, Germany). PBMCs were collected in conical tubes and washed in PBS (Gibco, Gaithersburg, MD, USA). Before staining, PBMCs were washed with FACS buffer (PBS, 1% bovine serum albumin [BSA] and 0.01% NaN3).
5
Peripheral Blood Mononuclear Cell Isolation
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Single-cell suspensions were obtained from peripheral blood. PBMCs were isolated from heparinised venous blood by density-gradient centrifugation over a Biocoll gradient solution (Biochrom AG, Berlin, Germany). The cells were washed with phosphate-buffered saline. Single cells were washed with FACS buffer (phosphate-buffered saline, 1% bovine serum albumin, 0.01% NaN3). The Abs used were fluorescein isothiocyanate anti-human CD103 (clone Ber-ACT8; eBioscience, San Diego, CA, USA), PE-cy7 anti-human CD45RO (clone UCHL1; eBioscience), antigen presenting cell (APC)-cy7 anti-human CD4 (clone OKT4; BioLegend, San Diego, CA, USA), and PE anti-human CD8 (clone RPA-T8; BD Pharmingen, Franklin Lakes, NJ, USA). Ab staining was performed for 30 min in darkness at room temperature. After washing with FACS buffer, at least 50,000 cells per sample were collected for analysis on a flow cytometer (BD FACS Aria™; Becton Dickinson, San Jose, CA, USA) and analysed in FlowJo™ software (Tree Star, Ashland, OR, USA).
6
Multiparametric Flow Cytometry Analysis
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We measured the frequencies of CD4+ T cells, CD8+ T cells, CD45RO+ memory cells, DCs, NK cells, monocytes, and chemokine receptors. Single-cell suspensions were obtained from peripheral blood. PBMCs were isolated from heparinised venous blood by density-gradient centrifugation over a Biocoll gradient solution (Biochrom AG). PBMCs were washed with phosphate-buffered saline. Single cells were washed with FACS buffer (phosphate-buffered saline, 1% bovine serum albumin, 0.01% NaN3). To detect DCs, we used a PerCP HLA-DR Ab (clone L243; BioLegend). To detect NK cells, PE-cy7 CD16 (clone CB16; eBioscience) and APC-CD56 (clone B159; BD Pharmingen) Abs were used. To detect monocytes, we used the APC-cy7 Ab CD14 (clone M5E2; BioLegend). To detect chemokine receptors, PerCP-cy5.5 anti-human CCR4 (clone 291H4; BioLegend), PE anti-human CCR6 (clone 11A9; BD Pharmingen), and APC anti-human CXCR3 (clone IC6; BD Pharmingen) Abs were used. Ab staining was performed for 30 min in darkness at room temperature. After washing with the FACS buffer, at least 50,000 cells per sample were collected for analysis on a flow cytometer (BD FACS Aria™; Becton Dickinson), and analysed in FlowJo™ software (Tree Star).
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