A total of three human BC cancer cell lines were used in this study, including T47D, SK-BR-3 and MDA-MB-231, which represent BC with a Luminal subtype, a HER2 ( +) subtype, and a TN subtype, respectively. All of these cell lines were provided by the Key Laboratory of Cancer Immunology and Biotherapy in our institution. The cells were regularly cultured in Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a 5% CO2 incubator. Human peripheral blood samples were obtained from healthy volunteer blood donors (Blood Center in our city) to isolate peripheral blood mononuclear cells (PBMCs) by centrifugation on Ficoll density gradients (GE Healthcare Life Sciences, Shanghai, China). Freshly obtained PBMCs were then incubated and activated in 10% FBS Roswell Park Memorial Institute (RPMI) 1640 mediums and used in the following transwell co-culture.
Ficoll density gradient
Manufactured by GE Healthcare
Sourced in Italy, United States, United Kingdom, China, Sweden
Ficoll density gradient is a laboratory technique used for the separation and purification of cells, organelles, and other biological macromolecules. It utilizes a solution of Ficoll, a high-molecular-weight, branched polysaccharide, to create a density gradient that allows the differential sedimentation of different components in a sample.
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Lab products found in correlation
Fetal bovine serum (fbs), by Euroclone (4 mentions)
Facsaria 2, by BD (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Cellquest software, by BD (2 mentions)
M csf, by R&D Systems (2 mentions)
Alpha mem, by Euroclone (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (2 mentions)
Ms column, by Miltenyi Biotec (2 mentions)
Cd34 microbead kit, by Miltenyi Biotec (2 mentions)
Paramagnetic macs beads, by Miltenyi Biotec (2 mentions)
Dnase, by Worthington (2 mentions)
Anti cd14 microbeads, by Miltenyi Biotec (2 mentions)
Hanks balanced salt solution (hbss), by Merck Group (2 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (2 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (2 mentions)
Dextran, by Merck Group (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Anti cd3 antibody, by Thermo Fisher Scientific (1 mentions)
Gt t551, by Takara Bio (1 mentions)
61 protocols using ficoll density gradient
1
Breast Cancer Cell Lines and PBMC Coculture
2
Isolation and Characterization of CIK Cells
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Human peripheral blood samples were obtained from healthy volunteer blood donors of blood (Tianjin Blood Center). PBMCs were isolated by centrifugation on Ficoll density gradients (GE Healthcare Life Sciences, Shanghai, China). To induce CIK cells, PBMCs were incubated in serum-free medium GT-T551 (Takara, Japan) containing 100 ng/mL anti-CD3 antibody (e-Bioscience, San Diego, USA), 100 U/mL rhIL-1α, and 1000 U/mL rhIFN-γ (R&D system, Inc, Minneapolis, MN) on day 1. Subsequently, 200 U/mL rhIL-2 was added to the medium on day 2, and the medium was regularly replaced with fresh IFN-γ- and IL-2-containing medium every 3–5 days. On day 14, cells were harvested and analyzed for the phenotypes of CIK cells by flow cytometric assay. Briefly, the phenotypes of induced CIK cells were detected through flow cytometry using a series of fluorescence-labeled monoclonal antibodies specific for CD3, CD4, CD8, CD25, CD127, CD16/CD56, CD45RA, CD45RO (eBioscience, San Diego, CA). Induced CIK cells (5 × 105) were incubated with these antibodies for 30 min on ice, and then cells were washed twice and analyzed on Fluorescence-Activated Cell Sorting (FACS) Aria I (BD Biosciences, San Diego, CA, USA) by using CellQuest software (BD Biosciences, San Diego, CA, USA).
3
Isolation and Activation of Macrophages
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The inflammasome reporter macrophages stably transduced with constructs for the expression of mCerulean or mCherry-tagged ASC have been described14 (link). Bone-marrow derived macrophages (BMDMs) were obtained by culturing bone marrow cells from 6- to 8-week old C57BL/6 mice in DMEM supplemented with 10% FBS, 10 μg/ml Ciprobay-500 and 40 ng/ml M-CSF (R&D Systems). Six days later, BMDMs were collected and plated. Immortalized BMDMs were cultured in DMEM supplemented with 10% FBS and 10 μg/ml Ciprobay-500. Human peripheral blood mononuclear cells (PBMCs) were purified from whole blood over Ficoll density gradients (GE Healthcare); erythrocytes were lysed in red cell lysis buffer (Miltenyi Biotec) and seeded in RPMI supplemented with 10% FBS and 10 μg/ml Ciprobay-500. The monocytic cell line THP1 was cultured in RPMI 1640 supplemented with 10% FBS and 10 μg/ml Ciprobay-500. For stimulation assays, cells were treated with 100 nM of phorbol 12-myristate 13-acetate (PMA) overnight, primed with 1 μg/ml of LPS for 2 h and further activated with 10 μM of nigericin for the time indicated in figure legends.
4
Isolation and Expansion of MSCs from BM
5
COVID-19 Patient Plasma and Immune Cell Analysis
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De-identified patient plasma and serum samples were obtained from discarded, clinically indicated collection of blood samples obtained from 382 patients admitted at ASST Spedali Civili Brescia, following positive nasopharyngeal swab and/or positive serology for SARS-CoV-2 infection. As described in the following sections and the main text, a subset of 60 unique patients, including 33 patients who had peripheral immune cells collected and 38 critically ill patients with deceased or recovery outcomes (for circulating protein/cytokine-based validation of the “critical juncture” concept in Figure 6 ) were analyzed in this study (i.e., 7 recovered and 4 deceased patients also had PBMCs collected). Ethical approval was obtained from the Comitato Etico Provinciale (NP 4000 – Studio CORONAlab), Brescia (Italy). Blood samples were processed to obtain serum and plasma. In addition, EDTA-blood samples were processed using Ficoll density gradient (GE Healthcare, Marlborough, MA) to obtain PBMCs, then washed twice in RPMI-1640; 5x106 PBMC were then resuspended in 1 mL of freezing medium consisting of RPMI plus 20% fetal calf serum and 10% DMSO, and then frozen at −80°C. Patient metadata is available in Tables S1 and S6 and deposited along with the sequencing data in GEO and code in Github listed above.
6
Isolation and Characterization of Human Mesenchymal Stromal Cells
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Bone marrow samples were obtained from healthy donors after written informed consent. Cells were separated on Ficoll density gradient (GE Healthcare, Italy), and the mononuclear cell fraction was collected and washed in PBS. We seeded 1 to 2.5 × 105 cells/cm2 in alpha-minimum essential medium (alpha-MEM) (EuroClone, Italy) containing 10% fetal bovine serum (FBS) (EuroClone, Italy), 100U/mL penicillin and 100 mg/mL streptomycin (EuroClone, Italy), 1× L-Glutamine (EuroClone, Italy), and 3 ng/mL basic fibroblast growth factor (bFGF) (Preprotech, United Kingdom). After 72 h, non-adherent cells were discarded and adherent cells were further cultivated to confluency and amplified at P1. Then, the medium was changed every 3 d and cells harvested when they reached 70%–80% confluence using Trypsin-EDTA (EuroClone, Italy) and routinely sub-cultured at 1:3 dilution.
We evaluated on MSCs the expression of markers recognized as one of the criteria to identify MSCs[20 (link)]. We verified by immunocytochemistry that MSCs expressed the surface antigens CD73, CD90 and CD105.
We evaluated on MSCs the expression of markers recognized as one of the criteria to identify MSCs[20 (link)]. We verified by immunocytochemistry that MSCs expressed the surface antigens CD73, CD90 and CD105.
7
PBMC Isolation for Immunologic Studies
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Blood samples for immunologic studies were collected at baseline from HCs and at baseline and 6 months after IFN-β-1a SC treatment from patients with RRMS. Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll density gradient (GE Healthcare Life Sciences, Pittsburgh, PA). CD4+ T cells and CD14+ monocytes were isolated from PBMCs using magnetic bead separation (Mylteni Biotech, San Diego, CA); purity was consistently >95%.
8
Mesenchymal Stem Cell Isolation and Culture
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Bone marrow was obtained from healthy donors (18 to 40 years of age) who provided written informed consent in accordance with the Declaration of Helsinki (1964) and Campania region Ethical Committee (nr 379/CE of Dec 16, 2016). We used plastic adherence and other minimal criteria suggested by the International Society for Cellular Therapy to identify MSCs containing a subpopulation of stem cell (Supplementary Files 1 2
9
Investigating Monocyte Immune Responses in Neurological Disorders
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Human peripheral blood mononuclear cells (PBMCs) were obtained using a Ficoll density gradient (GE Healthcare, Pasching, Austria) of buffy coats from healthy donors, NMOSD, and MS patients. Monocytes were purified using a CD14+ magnetic separation system (MACS, Miltenyi Biotec, Sunnyvale, CA, USA). The purity and viability of purified cells were assessed by flow cytometry and PI staining, respectively. All monocyte and PBMC cultures were performed in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (HyClone Laboratories, Logan, UT, USA) and 1% penicillin/streptomycin (GE Healthcare, Pasching, Austria).
For cytokine production and surface molecule analysis, CD14+ purified monocytes (5.0 × 105 cells/mL) were incubated for 24 h at 37 °C in complete medium. Monocytes were left unstimulated or were stimulated with recombinant human IFN-γ (100 ng/mL, R&D systems, Minneapolis, MN, USA), or recombinant human CD40L (1.0 μg/mL, Enzo Life Sciences, Farmingdale, NY, USA) or both, to mimic encounters with activated T cells. Supernatants were collected for cytokine detection, and cells were detached for flow cytometry analysis. For ICS, CD14+ purified monocytes (5 × 106 cells/mL) were cultured in complete medium, with or without CD40L, for 6 h.
For cytokine production and surface molecule analysis, CD14+ purified monocytes (5.0 × 105 cells/mL) were incubated for 24 h at 37 °C in complete medium. Monocytes were left unstimulated or were stimulated with recombinant human IFN-γ (100 ng/mL, R&D systems, Minneapolis, MN, USA), or recombinant human CD40L (1.0 μg/mL, Enzo Life Sciences, Farmingdale, NY, USA) or both, to mimic encounters with activated T cells. Supernatants were collected for cytokine detection, and cells were detached for flow cytometry analysis. For ICS, CD14+ purified monocytes (5 × 106 cells/mL) were cultured in complete medium, with or without CD40L, for 6 h.
10
Isolation and Activation of CD4+ T Effector Memory Cells
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We isolated peripheral blood mononuclear cells (PBMC) from whole blood using a Ficoll density gradient (GE Healthcare). We then isolated CD4+ effector memory T cells from PBMCs first by magnetic-activated cell sorting to enrich for CD4+ T cells, followed by fluorescent-activated cell sorting using labeled antibodies against CD45RA, CD45RO, and CD62L.
We stimulated CD4+ TEM cells by incubation with commercial anti-CD3/CD28 beads for 72 hours. For proliferation studies, we labeled cells with carboxyfluorescein diacetate succinimidyl ester (CFSE; eBioscience), and measured proliferation by dye dilution. Detailed isolation and purification methods are described inText S1 Figure S2A
We stimulated CD4+ TEM cells by incubation with commercial anti-CD3/CD28 beads for 72 hours. For proliferation studies, we labeled cells with carboxyfluorescein diacetate succinimidyl ester (CFSE; eBioscience), and measured proliferation by dye dilution. Detailed isolation and purification methods are described in
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