Neutrophils were isolated from whole blood using Hypaque Ficoll separation. Briefly, 1:1 PBS diluted blood was added onto the top of Ficoll (lymphocyte separation medium, MP Biomedicals), centrifuged at 800x g at room temperature for 20 minutes with the brake off. Neutrophils were retrieved from the bottom layer containing RBCs. After RBC lysis using ACK lysing buffer, cell pellets were washed twice in PBS. Neutrophil purity was routinely ~95% based on surface marker expression (CD16+, CD3−/CD19−) as determined by flow cytometry.
Ficoll
Manufactured by MP Biomedicals
Sourced in United States
Ficoll is a synthetic, high-molecular-weight, neutral, highly branched, and hydrophilic polysaccharide that can be used for density gradient centrifugation. It is commonly used for the isolation and purification of various cell types, including mononuclear cells, from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Merck Group (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (2 mentions)
Collagenase d, by Worthington (2 mentions)
Facsaria 2 flow cytometer, by BD (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Ack lysis buffer, by Quality Biological (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Rpmi8226, by American Type Culture Collection (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Mm 1s, by American Type Culture Collection (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Complete click s medium, by Irvine Scientific (1 mentions)
Anti human cd3, by BioLegend (1 mentions)
Rhil 2, by Thermo Fisher Scientific (1 mentions)
8 protocols using ficoll
1
Neutrophil Isolation from Whole Blood
2
Multicolor Flow Cytometry for Immune Cell Isolation
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Peripheral blood was separated into mononuclear cells and granulocytes via centrifugation over Ficoll (MP Biomedicals, Solon, OH); incubated with ammonium-chloride-potassium (ACK) lysis buffer (Quality Biological, Gaithersburg, MD) to remove red blood cells; and labeled with fluorphore-conjugated monoclonal antibodies against CD3, CD20, CD14, CD56, CD16, and CD33 (antibody clone and source information summarized in Table 2 ), allowing sorting of individual granulocyte, monocyte, T cell, and B cell lineages on a FACSAriaII flow cytometer (BD Biosciences, San Jose, CA). DNA was extracted from sorted cell pellets using the DNeasy Blood and Tissue Kit (QIAGEN, Valencia, CA).
Fluorochrome-Conjugated Antibodies
| Antigen | Antibody Clone | Conjugate Fluorophore | Company and Catalog Number |
|---|---|---|---|
| CD3 | SP34-2 | APC-Cy7 | BD Pharmingen (557757) |
| CD20 | 2H7 | PE-Cy5 | BD Pharmingen (555624) |
| CD14 | TUK4 | Pacific Blue | Invitrogen (MHCD1428) |
| CD16 | 3G8 | APC | BioLegend (302012) |
| CD56 | B159 | PE | BD Pharmingen (555516) |
| CD33 | AC104.3E3 | PE | Miltenyi Biotec (130-091-732) |
APC, allophycocyanin.
3
Multiple Myeloma Cell Line and Primary Cell Culture
4
Isolation and Characterization of Tumor-Infiltrating Lymphocytes
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Tumors were collected in ice-cold RPMI 1640 containing 2% FBS and minced into fine pieces, followed by digestion with 400 U/mL collagenase D (Worthington Biochemical Corporation, #LS004186) and 20 µg/mL DNase I (Sigma, #10104159001) at 37 °C for 40 min with periodic shaking. EDTA (Sigma, #1233508) was then added to the final concentration of 10 mM to stop digestion. Cell suspensions were filtered through 70 µM cell strainers, and TILs were obtained by collecting the cells in the interphase after Ficoll (MP Biomedicals, #091692254). Spleens were collected in ice-cold HBSS containing 2% FBS to prepare single-cell suspensions after lysis of red blood cells and filtering with 70 µM nylon mesh. Both TILs and splenocytes were resuspended in complete Click’s culture medium (Irvine Scientific, #9195-500 mL) for flow cytometric analyses. In some experiments, isolated TILs were cultured with 100 U/mL IL-2, with or without 1 μM Ruxo for 3 days and analyzed for FoxP3 expression and production of IFN-γ/TNF by flow cytometry, as described below.
5
Isolation and analysis of tumor-infiltrating lymphocytes
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Tumors were collected into ice-cold RPMI 1640 containing 2% FBS and minced into fine pieces on ice, followed by digestion with 400 U/mL collagenase D (Worthington Biochemical Corporation, #LS004186) and 20 μg/mL DNase I (Sigma, #10104159001) at 37 °C for 40 min with periodic mixing. EDTA (Sigma, #1233508) was then added to the final concentration of 10 mM to stop digestion. Cell suspensions were filtered through 70 μM cell strainers, and TILs were obtained by collecting the cells in the interphase after Ficoll (MP Biomedicals, #091692254) separation. Spleens and tumor-draining lymph nodes were collected in ice-cold HBSS containing 2% FBS to prepare single cell suspensions. Cells were filtered through 70 μM nylon mesh after lysis of red blood cells. TILs, dLNs and splenocytes were all re-suspended in complete Click’s medium (Irvine Scientific, #9195–500mL) for following staining and flow cytometric analyses.
6
Primary T cell Isolation and Expansion
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Primary T cells obtained from healthy consenting donors, 10 mL of blood was taken using a DG-veinset (VSET21) into LH Lithium Heparin tube (Greiner Bio-One, Austria, Kremsmünster). Blood was diluted in a 1:1 ration using PBS 2% FBS and loaded to a Ficoll (Mp-Bio, USA, CA) and separated by centrifugation at 400 × g for 30 min. Mononuclear cells were collected as the interphase, washed twice using PBS 2% FBS, counted and plated at 3.75 × 106/mL in 24-well plates in RPMI 10% human serum (Sigma, MO, USA), 300 U/mL rhIL2 (PeproTech) and 50 ng/mL of anti-human CD3 (BioLegend). After 48 h cells were harvested and re-plated in complete media without OKT3. Cells were kept in a humidified 5% CO2 incubator.
7
Isolation and Co-culture of PBMCs
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Blood samples from patients with AR (n = 22) were obtained from The First Affiliated Hospital, Sun Yat-sen University. This study was approved by The Ethics Committee of The First Affiliated Hospital, Sun Yat-sen University, and informed consents were obtained from all participants. Human buffy coats of volunteers were from Guangzhou Blood Center, exemption of written informed consent was approved.
Human peripheral blood mononuclear cells (PBMCs) from patients with AR or buffy coats of volunteers were isolated using Ficoll (MP Biomedicals, Santa Ana, Calif) density gradient centrifugation. PBMCs from buffy coats were used for the generation of DCs and the sort of ILC2s. PBMCs from patients with AR were co-cultured with DCs and DCs treated with sEVs.
Human peripheral blood mononuclear cells (PBMCs) from patients with AR or buffy coats of volunteers were isolated using Ficoll (MP Biomedicals, Santa Ana, Calif) density gradient centrifugation. PBMCs from buffy coats were used for the generation of DCs and the sort of ILC2s. PBMCs from patients with AR were co-cultured with DCs and DCs treated with sEVs.
8
Childhood Acute Lymphoblastic Leukemia Bone Marrow Isolation
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Subjects and cell isolation. This study was approved by the Ethics Committee of the Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University. Written informed consent was obtained from all the participants or their parents.
Bone marrow samples were collected from patients aged from 0 to 14 years who suffered from childhood acute lymphoblastic leukaemia (ALL) or a non-malignant blood disease. In total, 30 subjects with B-cell ALL (12 patients in the primary phase, 12 in the complete remission phase who were still undergoing treatment, 3 patients in the complete remission phase who had completed their treatment course and 3 in the relapse or non-remission phase) and 8 subjects with non-malignant blood disease were enrolled. The diagnoses of ALL were based on morphologic and flow cytometric analyses of the immunophenotype, and the stratification of ALL was based on the IC-BFM 2002 criteria for ALL. Bone marrow mononuclear cells (BMMCs) were isolated via Ficoll (MP Biomedicals, Santa Ana, CA, USA) density gradient centrifugation (14) .
Bone marrow samples were collected from patients aged from 0 to 14 years who suffered from childhood acute lymphoblastic leukaemia (ALL) or a non-malignant blood disease. In total, 30 subjects with B-cell ALL (12 patients in the primary phase, 12 in the complete remission phase who were still undergoing treatment, 3 patients in the complete remission phase who had completed their treatment course and 3 in the relapse or non-remission phase) and 8 subjects with non-malignant blood disease were enrolled. The diagnoses of ALL were based on morphologic and flow cytometric analyses of the immunophenotype, and the stratification of ALL was based on the IC-BFM 2002 criteria for ALL. Bone marrow mononuclear cells (BMMCs) were isolated via Ficoll (MP Biomedicals, Santa Ana, CA, USA) density gradient centrifugation (14) .
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