Single‐cell splenic suspensions were produced after collecting spleens from the mice followed by density gradient centrifugation supplied by Ficoll‐Paque (Nanjing Keygen Biotech). Single‐cell splenic suspensions were used to collect CD4+CD25+ Tregs and CD4+CD25− T cells by CD4+CD25+ Tregs isolation kits that contain a mix of 1‐ml monoclonal biotin‐conjugated antibodies to neutralize the CD8a, CD45R, CD11b, CD49b, and Ter‐119 cells of the mice under study, 2 ml microbeads to neutralize biotin, 1 ml of mouse CD25 antibodies conjugated with phycoeryanate, and 1 ml of anti‐ phycoeryanate microbeads supplied by Miltenyi Biotec GmbH) and a separator supplied by MiniMACStm (Miltenyi Biotec GmbH) as per the instructions of the manufacturer.
Minimacs
Manufactured by Miltenyi Biotec
Sourced in Germany, United States, Italy
The MiniMACS is a compact and easy-to-use magnetic cell separation system from Miltenyi Biotec. It is designed for the separation and enrichment of target cells from heterogeneous cell populations using magnetic microbeads.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd19 microbeads, by Miltenyi Biotec (4 mentions)
70 mm filter, by BD (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Hepes, by Solarbio (2 mentions)
Type iv1 collagenase, by Merck Group (2 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Merck Group (2 mentions)
Dispase, by Merck Group (2 mentions)
Hanks balanced salt solution (hbss), by Solarbio (2 mentions)
Mouse tcrγ δ t cell isolation kit, by Miltenyi Biotec (2 mentions)
Density gradient centrifugation, by Organon (2 mentions)
Heparin, by Thermo Fisher Scientific (2 mentions)
Lineage cell depletion kit, by Miltenyi Biotec (2 mentions)
Pharm lyse lysing solution, by BD (2 mentions)
Macs cd14 microbeads, by Miltenyi Biotec (2 mentions)
Magna lyser green beads, by Roche (2 mentions)
Magna pure compact rna isolation kit, by Roche (2 mentions)
Rneasy lipid tissue mini kit, by Qiagen (2 mentions)
Cd11b positive selection, by Miltenyi Biotec (2 mentions)
Vacutainer cpt tube, by BD (2 mentions)
51 protocols using minimacs
1
Isolation of Regulatory T Cells from Murine Spleens
2
Isolation and Purification of Colonic Lymphocytes and γδT Cells
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The colons of mice were incubated in a 37°C water bath in cell dissociation solution made with HBSS and HEPES (Solarbio) to strip the epithelial cells. Supernatant was discarded, and colonic tissue was then incubated in a digestion cocktail containing HBSS, FCS (10%, Gibco), type IV1 collagenase (1 mg/ml), DNaseI (0.5 mg/ml), and dispase (0.5 mg/ml, all from Sigma-Aldrich) in a 37°C water bath. After that, the digested tissue was processed through a 70 mm filter (Falcon) and washed before lymphocytes were separated by the methods used previously (20 (link), 24 (link)). γδT cells were purified from the spleens of mice using the Mouse TCRγδ+ T-cell Isolation Kit and the miniMACSTM (Miltenyi Biotec, Germany).
3
Purification of Murine Colonic Lymphocytes
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The colons of mice were incubated in a 37°C water bath in cell dissociation solution made with HBSS and HEPES (Solarbio) to strip the epithelial cells. Supernatant was discarded and colonic tissue was then incubated in a digestion cocktail containing HBSS, FCS (10%, Gibco), type IV1 collagenase (1 mg/ml), DNaseI (0.5 mg/ml), and dispase (0.5 mg/ml, all from Sigma–Aldrich) in a 37°C water bath. After that, the digested tissue was processed through a 70 mm filter (Falcon) and washed before lymphocytes were separated by the methods used previously. γδT cells were then purified from the spleens of mice using the Mouse TCRγδ + T-cell Isolation Kit and the miniMACSTM (MiltenyiBiotec, Germany).
4
Isolation and Analysis of CD34+ Cells
5
Isolation and Analysis of CD34+ Cells
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Mononuclear cells from peripheral blood or bone marrow were isolated by density gradient centrifugation (OrganonTeknika, Durham, NC) and cryopreserved in RPMI1640 supplemented with 20% fetal calf serum and 10% dimethyl sulfoxide. After thawing, CD34+ cells were selected by immunomagnetic beads (MiniMACS, Miltenyi Biotech, Auburn, CA). Sample date varied between the subjects, but all samples from relapsed patients were collected when the concurrent peripheral blood showed either positive BCR-ABL or hematological relapse after the diagnosis of relapse (median time from diagnosis of relapse to sample collection was 386 days). Six patients who were on TKI alone and never received allo-SCT, served as controls (Supplementary Table 1 ).
6
T Cell Isolation and Transfer
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CD4+ T cells and/or CD8+ T cells were isolated from the spleens of B6 and/or BALB/c mice using microbead-conjugated antibodies (MiniMACS™; Miltenyi Biotec; #130-049-201, and #130-049-401). The purity was consistently > 92 %. T cells were cultured in 96-well U-bottom plates (Corning, Corning, NY, USA; #3799), complete RPMI-1640 medium containing human IL-2 (200 IU/mL), and activated DCs. The ratio of T cells to DCs ranged from 2:1 to 4:1. T cells were cultured for three days at 37 °C with 5 % CO2, and then collected and injected into the experimental mice. The number of injected T cells was 2 × 106/mouse.
7
Isolation and Characterization of CD206+ Tumor Macrophages
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CD206+ macrophages were isolated from single-cell suspensions from tumor tissue using CD206-APC primary antibody and anti-APC magnetic beads with miniMACS (MiltenyiBiotec, Auburn, CA, USA) according to manufacturer's protocol [6] . The CD206+ macrophage fraction contained >95% macrophages, as confirmed by flow cytometry.
mRNA isolation and reverse transcription-PCR (RT-PCR), semiquantitative and quantitative real-time PCR Total RNA was isolated and RT-PCR performed as previously described [6] .
mRNA isolation and reverse transcription-PCR (RT-PCR), semiquantitative and quantitative real-time PCR Total RNA was isolated and RT-PCR performed as previously described [6] .
8
Isolation and Differentiation of Monocytes
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Blood samples from 4 healthy blood donors from the Transfusion Center at the Sapienza University of Rome were used to obtain peripheral blood mononuclear cells (PBMCs). The study was conducted in accordance with the Helsinki Declaration of 1975 and 1983.
Monocytes were obtained from PBMCs, as described previously [18 (link)]. In brief, PBMCs were isolated by density gradient (Lympholyte, Cedarlane, Oxford, UK). CD14+ monocytes were purified by incubating PBMCs with anti-CD14-coated microbeads, followed by sorting with a magnetic device (MiniMacs, Miltenyi Biotec). Monocytes were induced to differentiate for 6 days in cell culture dishes (100 mm) (BD-Biosciences, San Diego, CA), in the presence of either rhGM-CSF (10 ng/mL) to obtain M1 macrophages or rhM-CSF (10 ng/mL) to obtain M2 ones. Cells were cultured at 8 × 105 cells/mL in RPMI 1640—supplemented with 1% nonessential amino acids, 1% sodium pyruvate, 50 U/mL penicillin, 50 μg/mL streptomycin, 5 × 10−5 M 2-mercaptoethanol, and 10% FBS.
Monocytes were obtained from PBMCs, as described previously [18 (link)]. In brief, PBMCs were isolated by density gradient (Lympholyte, Cedarlane, Oxford, UK). CD14+ monocytes were purified by incubating PBMCs with anti-CD14-coated microbeads, followed by sorting with a magnetic device (MiniMacs, Miltenyi Biotec). Monocytes were induced to differentiate for 6 days in cell culture dishes (100 mm) (BD-Biosciences, San Diego, CA), in the presence of either rhGM-CSF (10 ng/mL) to obtain M1 macrophages or rhM-CSF (10 ng/mL) to obtain M2 ones. Cells were cultured at 8 × 105 cells/mL in RPMI 1640—supplemented with 1% nonessential amino acids, 1% sodium pyruvate, 50 U/mL penicillin, 50 μg/mL streptomycin, 5 × 10−5 M 2-mercaptoethanol, and 10% FBS.
9
Quantifying B Cell Activation Markers
10
Isolation of Bone Marrow Stem Cells
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Bone marrow (BM) was collected from ALS patients after obtaining their informed consent. Each time, 40-50 mL of BM samples were harvested in local anesthesia from the posterior iliac crest and subsequently resuspended in collecting medium (PBS, pH 7.2) and heparin (20 U/mL; Gibco, USA). The collected BM was lysed in BD PharmLyse Lysing Solution (BD Biosciences, San Jose, USA) for 15 min at room temperature in the dark and washed twice in phosphate-buffered saline (PBS). The procedure was performed in compliance with the manufacturer's protocol, as previously described 11 . The obtained suspension of BM nucleated cells (NCs) was subjected to immunomagnetic separation (MiniMACS, Miltenyi Biotec, Auburn, USA). LIN- stem/progenitor cells (mean 8.98 ± 5.77×106) were isolated from non-separated NCs using immunomagnetic isolation and a Lineage Cell Depletion Kit (Miltenyi Biotec, Auburn, USA), as previously described 20 (link). Before administration, the isolated cells were kept in 2 mL of sterile PBS.
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