Human monocytes were isolated by Ficoll-plaque plus (GE healthcare) centrifugation of human blood followed by the collection of monocytes attached to the plastic surface after 4 hours culture. Briefly, blood from donors were diluted in Hank’s buffer to a 1:1 solution. The blood solution was placed over half volume of Ficoll-plaque plus (GE healthcare) and centrifuged at 1,600rpm for 30 minutes. The middle layer of PBMCs were collected and cultured in RPMI/1640 medium supplemented with 10% FBS. 4 hours after culture, the floating cells were washed away with PBS. The adherent monocytes were trypsinized and subjected to migration or invasion assays. All studies related to human subjects were approved by the Institutional Review Board of the Cleveland Clinic. Informed consent was obtained from all subjects. For transwell assays, 5×104 human monocytes/macrophages were cultured in the upper chamber while recombinant POSTN protein (0.5 μg/ml) in RPMI/1640 media with 0.1% BSA was added in the lower chamber. Cells were allowed to migrate or invade for 24h and the migrated cells in the bottom of the well were fixed and stained.
Ficoll plaque plus
Manufactured by GE Healthcare
Sourced in United States, Sweden, United Kingdom
Ficoll-Plaque Plus is a density gradient medium used for the separation and purification of cells, organelles, and other biological particles. It is based on a synthetic high-molecular-weight polysaccharide that allows efficient separation of different cell types and subcellular components during centrifugation. The product's core function is to facilitate the isolation and enrichment of specific cell populations or cellular components from complex biological samples.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (3 mentions)
Rbc lysis buffer, by Thermo Fisher Scientific (2 mentions)
Cell counting kit 8 reagent, by Dojindo Laboratories (2 mentions)
Immunomagnetic selection, by Miltenyi Biotec (2 mentions)
Magnetic beads prior, by STEMCELL (2 mentions)
Tri reagent, by Merck Group (2 mentions)
Facsaria flow cytometer, by BD (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Bend 3 atcc crl 2299, by American Type Culture Collection (1 mentions)
Hyclone, by GE Healthcare (1 mentions)
Penicillin streptomycin, by GE Healthcare (1 mentions)
Raw264, by American Type Culture Collection (1 mentions)
Dulbecco modified eagle medium (dmem), by Welgene (1 mentions)
Leucosep tube, by Greiner (1 mentions)
K2edta, by BD (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
78 protocols using ficoll plaque plus
1
Monocyte Isolation and Migration Assay
2
Monocyte Isolation and Migration Assay
3
Isolation and Culture of Mouse and Rat Immune Cells
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The mouse brain endothelial cell line bEnd.3 (ATCC CRL-2299) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and the mouse macrophage cell line, RAW 264.7, kindly provided from by Professor Namhyun Jung from Korea University. All cells were cultured in Dulbecco’s modified eagle medium (DMEM; Welgene, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS; HyClone, GE Healthcare Life Sciences, Logan, UT, USA) and 1% penicillin/streptomycin (HyClone, GE Healthcare Life Sciences, Logan, UT, USA) at 37 °C in a 5% CO2 humidified atmosphere.
Rat peripheral blood mononuclear cells were isolated from heparinized blood obtained from normal Sprague–Dawley (SD) rats via density gradient centrifugation (Ficoll-Plaque Plus, GE Health Care Life Sciences, Pittsburgh, PA, USA) according to the manufacturer’s instruction. Differentiation into macrophages was induced by a recombinant rat macrophage colony-stimulating factor (15 ng/mL; Peprotech, Rocky Hill, NJ, USA) with 1% penicillin/streptomycin (HyClone, GE Healthcare Life Sciences, Logan, UT, USA) and 10% heat-inactivated FBS (HyClone, GE Healthcare Life Sciences, Logan, UT, USA) for 7 days.
Rat peripheral blood mononuclear cells were isolated from heparinized blood obtained from normal Sprague–Dawley (SD) rats via density gradient centrifugation (Ficoll-Plaque Plus, GE Health Care Life Sciences, Pittsburgh, PA, USA) according to the manufacturer’s instruction. Differentiation into macrophages was induced by a recombinant rat macrophage colony-stimulating factor (15 ng/mL; Peprotech, Rocky Hill, NJ, USA) with 1% penicillin/streptomycin (HyClone, GE Healthcare Life Sciences, Logan, UT, USA) and 10% heat-inactivated FBS (HyClone, GE Healthcare Life Sciences, Logan, UT, USA) for 7 days.
4
Serum and PBMC Isolation Protocol
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Blood was collected at the study day for serum storage (10 mL tubes; cat.no. 367895, BD) or for PBMC isolation (K2‐EDTA; 4 x 10mL; cat.no. 367525, BD). Serum tubes were left at room temperature for at least 30 min before centrifugation at 2000 x g 10 min at room temperature. Serum was aliquoted and stored at ‐80°C. PBMCs were isolated within 6 hours using 50 ml Leucosep tubes (227290, Greiner Bio-One) filled with Ficoll plaque plus (17-1440-02, GE Healthcare Life Sciences) according to manufacturer’s protocol. Remaining PBMCs were cryopreserved and stored in liquid nitrogen, protocol: dx.doi.org/10.17504/protocols.io.87phzmn .
5
Isolation and Storage of Plasma and PBMCs
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Samples of plasma and PBMCs have been isolated by whole blood using Ficoll Plaque Plus (GE Healthcare, Little Chalfont, UK) according to manufacturer instructions. Isolated PBMCs have been suspended in 1mL of Trizol (Ambion, Waltham, MA, USA) and stored at -80°C until further analysis. Plasma samples were centrifuged for 10’ at 16,000 x g in order to remove additional nucleic acids attached to cell debris and then stored at -80°C until further analysis.
6
Isolation of PBMCs from Blood
7
Tumor Immune Landscape in GIST
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Tumor specimens and matched peripheral blood were obtained from patients with GIST who underwent surgery at our institution and consented to a protocol approved by the Institutional Review Board at Memorial Sloan-Kettering. Blood was drawn before surgical incision, and peripheral blood mononuclear cells were isolated by density centrifugation over Ficoll-Plaque PLUS (GE Healthcare). Tumor tissue was sectioned and digested in 5 mg/ml collagenase IV (Sigma-Aldrich) and DNase I (0.5 mg/ml; Roche Diagnostics) in HBSS for 30 min while shaking at 37°C. After procurement, all processed cells were immediately analyzed with flow cytometry.
Protein chunks from human GIST tumors were flash frozen in liquid nitrogen and stored at −80°C until use. Total RNA was isolated using the RNAeasy Plus Mini Kit (Qiagen). Quality of RNA was ensured before labeling by analyzing 20 ng of each sample using the RNA 6000 NanoAssay and a Bioanalyzer 2100 (Agilent). High-throughput RNaseq was performed by the Integrated Genomics Core laboratory of the Sloan Kettering Institute using the Illumina platform and normalized using the software package DESeq. Cytolytic score was calculated as the square root of the product of granzyme A (GZMA) and perforin (PRF1) expression.
Protein chunks from human GIST tumors were flash frozen in liquid nitrogen and stored at −80°C until use. Total RNA was isolated using the RNAeasy Plus Mini Kit (Qiagen). Quality of RNA was ensured before labeling by analyzing 20 ng of each sample using the RNA 6000 NanoAssay and a Bioanalyzer 2100 (Agilent). High-throughput RNaseq was performed by the Integrated Genomics Core laboratory of the Sloan Kettering Institute using the Illumina platform and normalized using the software package DESeq. Cytolytic score was calculated as the square root of the product of granzyme A (GZMA) and perforin (PRF1) expression.
8
Cryopreservation of Healthy Human PBMCs
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Peripheral blood samples were obtained from healthy anonymous volunteers of the Region H blood bank (Copenhagen, Denmark) under informed consent, according to the protocol H-D-2008-113 for research use approved by the Danish Scientific Ethical Committee Region Hovedstaden (Legislative Order No. 402 of May 28th 2008). The donors were HLA-typed at HistoGenetics LLC using next generation sequencing (NGS) technology by the means of Illumnia MiSeq (Ossining, NY, USA). The human blood samples (buffy coat from 450ml blood) were processed as followed: PBMCs were purified by Ficoll-Plaque Plus (GE Healthcare, Uppsala, Sweden) density centrifugation. Red blood cells were lysed using RBC lysis buffer (eBioscience, San Diego, USA) and the PBMCs were washed twice in PBS. Average yield was 2-6x108 cells (Nucleocounter NC-200, Sartorius) and viability was 95.4%. Cells were frozen in CLTC-ABC freezing media (C.T.L), according to their protocol, at approximate 30x106cells/ml in CoolCell containers (Biocision).
9
Isolation of Human Eosinophils
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Human polymorphonuclear cells were separated from heparinized venous blood collected from healthy donors by using Ficoll-Plaque Plus (GE Healthcare Japan, Tokyo, Japan). Eosinophils were isolated from polymorphonuclear cells by negative selection using the Human Eosinophil Isolation kit (Miltenyi Biotec, Tokyo, Japan).
10
Oxaliplatin and S-1 Combination Chemotherapy
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Oxaliplatin and S-1 were obtained from Zhongda Hospital. For patients, oxaliplatin (85 mg/m2) was administered intravenously on day 1. S-1 (80 mg/(m2•day)) was administered orally twice daily for 14 days. All patients were then allowed 1 week of rest before the next cycle. Physical examination and blood analysis were performed at each cycle during chemotherapy and six cycles were administered to all patients.
Peripheral blood samples were obtained from patients pre- and post-operative and before and after the first or sixth cycle of chemotherapy. Peripheral blood samples from HVs were used as controls. Peripheral blood mononuclear cells (PBMC) were isolated with Ficoll plaque plus (#17144002, GE) using density gradient centrifugation within 2 h of sample collection. CD8+ TIL Cells were isolated from GC tissues using a human CD8+ T cell isolation kit (#17953, Stemcell) following the manufacturer’s instructions.
Peripheral blood samples were obtained from patients pre- and post-operative and before and after the first or sixth cycle of chemotherapy. Peripheral blood samples from HVs were used as controls. Peripheral blood mononuclear cells (PBMC) were isolated with Ficoll plaque plus (#17144002, GE) using density gradient centrifugation within 2 h of sample collection. CD8+ TIL Cells were isolated from GC tissues using a human CD8+ T cell isolation kit (#17953, Stemcell) following the manufacturer’s instructions.
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