Six-milliliter peripheral blood samples were prepared with EDTA-K2 tubes, and PBMCs were isolated by centrifugation through a Histopaque density gradient (H8889; Sigma). Next, PBMCs were stimulated with culture supernatants of oHSV2 or oHSV2-aPD1-infected or mock-infected A549 cells (filtered by 0.22-μm filters), 1 μg/mL ionomycin (BioGems, USA), and 50 ng/mL PMA (BioGems, USA). After being cultured in a 37°C, 5% CO2 incubator for 36 h, supernatants were collected for analysis of IFN-γ and IL-2 levels with human IFN-γ Pre-Coated ELISA Kit (BGK01579; BioGems) and human IL-2 Pre-Coated ELISA Kit (BGK60568; BioGems). Every sample was tested with a duplicate. All PBMCs from patients with advanced tumors were obtained after informed consent, and the collection of human specimens obtained approval from the Ethics Committee of National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College.
Histopaque density gradient
Manufactured by Merck Group
Sourced in Germany, United States
Histopaque density gradient is a laboratory product used for the separation and isolation of mononuclear cells from whole blood. It is a sterile, endotoxin-tested solution of polysucrose and sodium diatrizoate, adjusted to a density of 1.077 g/mL. When centrifuged, the Histopaque solution forms a density gradient that allows the specific isolation of the mononuclear cell layer.
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Lab products found in correlation
Genome wide snowball array, by Thermo Fisher Scientific (2 mentions)
Qiazol reagent, by Qiagen (2 mentions)
C57b6 l mice, by Daehan Biolink (2 mentions)
Ionomycin, by Biogems (1 mentions)
Phorbol myristate acetate (pma), by Biogems (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
1640 medium, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Facsaria 2, by BD (1 mentions)
Trap staining solution, by Merck Group (1 mentions)
Ambion wt expression kit, by Thermo Fisher Scientific (1 mentions)
Wt terminal labeling kit, by Thermo Fisher Scientific (1 mentions)
Gcos 1, by Thermo Fisher Scientific (1 mentions)
Cell strainer, by BD (1 mentions)
Trypan blue exclusion assay, by Merck Group (1 mentions)
Rpmi 1640 culture medium, by Thermo Fisher Scientific (1 mentions)
Anti cd14 clone hcd14, by BioLegend (1 mentions)
Anti cd49d clone 9f10, by BioLegend (1 mentions)
Anti cd11b clone m1 70, by BioLegend (1 mentions)
Cryostor cs5, by STEMCELL (1 mentions)
19 protocols using histopaque density gradient
1
PBMC Cytokine Secretion Assay for Oncolytic Virus
2
Gal-9 Secretion from Activated PBMCs
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PBMCs were isolated by centrifugation on a Histopaque density gradient (Sigma-Aldrich, St. Louis, MO, United States). The isolated PBMCs were seeded into 96-well plates at 5 × 105 cells/mL in Roswell Park Memorial Institute 1640 medium (Gibco, Grand Island, NY, United States) supplemented with 10% fetal bovine serum (Gibco) and 100 U/μg/mL penicillin/streptomycin (Gibco) at 37°C and 5% CO2 for 48 h. The supernatant was collected by centrifugation. An ELISA kit was used to determine Gal-9 levels in the supernatant, as described.
3
Immunophenotyping of OT-I T-Cells in Tumor Tissue
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For detecting chemokine receptor expression on splenocytes harvested from OT-I mice, the cells were cultured overnight in RPMI 1640 complete medium containing 300 U/ml of IL2 before they were doubly stained with anti-mouse CCR2, CXCR3 and CCR5 antibodies, in combination with either anti-CD4 or anti-CD8 antibodies, for 30 min at 4° C. Cells were then analyzed with BD FACSAria II (BD Biosciences, San Jose, CA).
To analyze the T-cell subsets of OT-I cells within tumor tissues, tumors were collected from the mice that had been used for the imaging experiment (after the termination of the experiment). The excised tumors were placed in dissociation buffer (100 U/ml collagenase type I and 100 μg/ml DNase in RPMI) for 30 min at 37 °C. The dissociated tumor tissues were then filtered through a 40 micron filter and washed 3X with PBS containing 2% fetal bovine serum (FBS) before they were loaded to Histopaque density gradient (Sigma-Aldrich, St. Louis, MO) and sedimented at 400 X g for 30 min. The interphase layer that was enriched with lymphocytes and monocytes were collected and counted. Cells were then stained with anti-mouse CD4 and CD8 antibodies alone or doubly stained for CD4-CD62L and CD8-CD62L for 30 min at 4° C, after which, they were incubated with anti-mouse CD16/32 antibody 30 min to block no-specific Fc receptor binding. The cell staining was analyzed with BD FACSAria II.
To analyze the T-cell subsets of OT-I cells within tumor tissues, tumors were collected from the mice that had been used for the imaging experiment (after the termination of the experiment). The excised tumors were placed in dissociation buffer (100 U/ml collagenase type I and 100 μg/ml DNase in RPMI) for 30 min at 37 °C. The dissociated tumor tissues were then filtered through a 40 micron filter and washed 3X with PBS containing 2% fetal bovine serum (FBS) before they were loaded to Histopaque density gradient (Sigma-Aldrich, St. Louis, MO) and sedimented at 400 X g for 30 min. The interphase layer that was enriched with lymphocytes and monocytes were collected and counted. Cells were then stained with anti-mouse CD4 and CD8 antibodies alone or doubly stained for CD4-CD62L and CD8-CD62L for 30 min at 4° C, after which, they were incubated with anti-mouse CD16/32 antibody 30 min to block no-specific Fc receptor binding. The cell staining was analyzed with BD FACSAria II.
4
Rabbit Bone Marrow MNC Isolation
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MNCs were harvested from the rabbits at day 21 after femoral artery removal. Overall, 5 mL of bone marrow was aspirated under anesthesia from right iliac crest through an infant aspiration needle (Terumo). The MNC fraction was isolated from aspirated bone marrow by centrifugation in Histopaque density gradient (Sigma-Aldrich), as described previously.13 (link)
5
Porcine Blood Analysis Protocol
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An aliquot of the collected whole blood was used for blood cell counts using an ABX Pentra 60 device (HORIBA ABX SAS, Montpellier, France). Plasma was prepared by centrifugation for 20 min at 4°C and 2000g and stored at −80°C until use. PBMCs were prepared via centrifugation on a Histopaque density gradient (Sigma-Aldrich, Taufkirchen, Germany). PBMCs isolated from slaughter blood were cryopreserved for subsequent cell culture analysis in a freezing medium consisting of 90% fetal bovine serum (FBS; PAA, Cölbe, Germany) and 10% DMSO.
Plasma ACTH, cortisol and aldosterone levels were measured in duplicate using commercially available enzyme-linked immunosorbent assays according to the manufacturer's recommendations (DRG, Marburg, Germany). The assays for ACTH and cortisol were validated for porcine plasma previously ([46 (link)] and [17 (link)], respectively). The intra- and inter-assay coefficients of variation of the aldosterone assay were both less than 4%.
Plasma levels of glucose, total cholesterol, triglycerides and blood urea nitrogen were measured using a Fuji DriChem 4000i clinical chemistry analyzer (Scil, Viernheim, Germany).
Plasma ACTH, cortisol and aldosterone levels were measured in duplicate using commercially available enzyme-linked immunosorbent assays according to the manufacturer's recommendations (DRG, Marburg, Germany). The assays for ACTH and cortisol were validated for porcine plasma previously ([46 (link)] and [17 (link)], respectively). The intra- and inter-assay coefficients of variation of the aldosterone assay were both less than 4%.
Plasma levels of glucose, total cholesterol, triglycerides and blood urea nitrogen were measured using a Fuji DriChem 4000i clinical chemistry analyzer (Scil, Viernheim, Germany).
6
Gene Expression Analysis of Porcine PBMCs
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For expression analysis a subset of animals was selected aiming for a balanced design in terms of gender, batch and ancestry. Accordingly, twelve castrated males and twelve females per coping group were selected for subsequent analyses, producing 48 individual samples per sampling time point. The PBMCs were isolated from 5 mL blood by centrifugation on a Histopaque density gradient (Sigma-Aldrich, Taufkirchen, Germany), then stored at -80°C. Total RNA was isolated using Qiazol reagent per manufacturer’s directions (Qiagen, Hilden, Germany). Quantification and purification were performed as previously described [24 (link)]. All RNA was stored at -80°C until downstream analyses were performed. For the microarray experiments, individual samples (n = 192) were hybridized on genome-wide snowball arrays (Affymetrix, Santa Clara, CA, USA), a platform invented for genome-wide analyses of the pig transcriptome [25 (link)]. Processing was performed as previously described [24 (link)]. Raw data have been deposited in a MIAME-compliant database, the National Center for Biotechnology Information Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo ) (accession numbers: GSE55418).
7
Isolation and Characterization of Human MSCs
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A 5-ml volume of bone marrow was obtained from four human patients. Mononuclear
cells were isolated using a Histopaque density gradient (Sigma-Aldrich, São
Paulo, SP, Brazil) of 1,077 g/ml. MSC culture was performed as described by
Rebelatto et al.15 . The remaining MSCs were cultured until the number of cells was
sufficient for transplantation, which occurred between passages 3 and 5. At that
time, cell characterization and viability assays were performed by flow
cytometry.
cells were isolated using a Histopaque density gradient (Sigma-Aldrich, São
Paulo, SP, Brazil) of 1,077 g/ml. MSC culture was performed as described by
Rebelatto et al.15 . The remaining MSCs were cultured until the number of cells was
sufficient for transplantation, which occurred between passages 3 and 5. At that
time, cell characterization and viability assays were performed by flow
cytometry.
8
Osteoclast Differentiation Assay
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Osteoclast differentiation was performed as previously described [18 (link)]. Bone marrow cells obtained from 6- to 8-week-old C57B6/L mice (Dae Han Bio Link, Chungbuk, Korea) were incubated in α-minimal essential medium (α-MEM) containing 10% fetal bovine serum (FBS). After 24 h, non-adherent cells were centrifuged on a Histopaque density gradient (Sigma-Aldrich, St. Louis, MO, USA) and cultured in α-MEM supplemented with 10% FBS and M-CSF (30 ng/mL) for 3 days to obtain bone marrow macrophages (BMMs). BMMs were cultured with RANKL (20 ng/mL) and M-CSF (10 ng/mL) in the absence or presence of 1 μM or 5 μM OCLI-023 for 4 days. Then, the cells were stained with a tartrate-resistant acid phosphatase (TRAP)-staining solution prepared following the manufacturer’s instructions (Sigma-Aldrich). TRAP-positive multinucleated cells (MNCs), having three or more nuclei, were counted as osteoclast-like cells.
9
Transcriptome Analysis of Porcine PBMC
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Samples obtained at 28 dpn and 63 dpn were used to isolate peripheral blood mononuclear cells (PBMC) from 5 ml of blood by centrifugation on a Histopaque density gradient (Sigma-Aldrich, Taufkirchen, Germany), and then stored at −80°C. Total RNA was isolated using Qiazol reagent per the manufacturer's directions (Qiagen, Hilden, Germany). Quantification and purification were performed as previously described (52 (link)). All RNA samples were stored at −80°C until transcriptome profiling was performed. Individual RNA samples were transcribed to DNA using the Ambion WT expression kit (Ambion, Austin, TX). DNA preparations were fragmented and labeled using the WT terminal labeling kit (Affymetrix, Santa Clara, CA). Subsequently, preparations were hybridized on genome-wide “snowball” arrays (Affymetrix, Santa Clara, CA), a platform invented for genome-wide analyses of the pig transcriptome (23 (link)). Raw data were generated using Affymetrix GCOS 1.1.1 software and were deposited in a MIAME-compliant database (19 (link)), the National Center for Biotechnology Information Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo; accession number: GSE66308 ).
10
Isolation of Mononuclear Cells
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Single cell suspensions from spleens were obtained by direct mechanical disruption in RPMI 1640 through a 40 μm mesh (Cell Strainer, BD). Thymuses were cut into very small pieces and mechanically disrupted by pressing with a syringe plunger, in RPMI 1640. Then, cellular suspensions were passed through a 40 μm mesh (Cell Strainer, BD). Mononuclear cells were isolated from both suspensions by centrifugation over Histopaque density gradient (1.077 g/mL; Sigma, St. Louis, MO, USA) at 400 × g for 30 min and room temperature. Cells were isolated from the interface, washed, and live cells were counted using trypan blue exclusion.
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