Blood from healthy individuals was collected from the antecubital vein into blood collection tubes containing sodium citrate. All study participants signed a written informed consent. Peripheral blood mononuclear cells (PBMCs) were separated from blood on a Ficoll-Paque (GE Healthcare) density gradient using LeucoSep separation tubes (Greiner), following centrifugation at 800 g for 15 min at room temperature without brake. Monocytes were negatively selected from PBMCs using Classical Monocyte Isolation Kit, LS Columns, and MidiMACS™ Separators (all from Miltenyi) according to the manufacturer’s instructions. Isolated monocytes were cultured in RPMI 1640, GlutaMAX™ Supplement (Thermo Fisher Scientific) media supplemented with 10% premium grade fetal bovine serum (P-FBS) with low endotoxin (Biowest) and 1:200 (volume/volume) penicillin–streptomycin solution (Sigma).
Midimacs separator
Manufactured by Miltenyi Biotec
Sourced in Germany, United States, Morocco
The MidiMACS Separator is a benchtop magnetic separation device designed for the efficient separation and isolation of magnetically labeled cells. The core function of the MidiMACS Separator is to provide a controlled magnetic field that allows for the effective separation of target cells from a heterogeneous cell suspension.
Automatically generated - may contain errors
Lab products found in correlation
Ls column, by Miltenyi Biotec (29 mentions)
Penicillin, by Thermo Fisher Scientific (12 mentions)
Anti biotin microbead, by Miltenyi Biotec (7 mentions)
L glutamine, by Thermo Fisher Scientific (7 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (6 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (5 mentions)
Ld column, by Miltenyi Biotec (5 mentions)
Mouse neutrophil isolation kit, by Miltenyi Biotec (4 mentions)
Ficoll paque plus, by GE Healthcare (4 mentions)
Cd14 microbead, by Miltenyi Biotec (4 mentions)
Macs ls column, by Miltenyi Biotec (4 mentions)
Cd45 microbeads, by Miltenyi Biotec (3 mentions)
M csf, by Thermo Fisher Scientific (3 mentions)
Anti pe microbeads, by Miltenyi Biotec (3 mentions)
Rpmi 1640 medium, by Atlanta Biologicals (3 mentions)
Mouse cell removal beads, by Miltenyi Biotec (3 mentions)
Pan t cell isolation kit 2, by Miltenyi Biotec (3 mentions)
Macs multistand, by Miltenyi Biotec (3 mentions)
Exoquick tc exosome isolation reagent, by System Biosciences (3 mentions)
Ficoll paque, by GE Healthcare (2 mentions)
106 protocols using midimacs separator
1
Isolation and Culture of Human Monocytes
2
Isolation and Expansion of NG2 Cells
3
Immune Cell Sorting in Injured Muscle
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For sorting of CD45+CD3+ T cells, CD45+CD11b+ monocytes, and CD45− muscle resident cells in injured muscle, tissues were minced and digested to single-cell suspension, then cells were labeled and sorted by Beckman Coulter MoFloTM XDP. In some experiments for the sorting of CD11b+ cells, CD11b microbeads, LS column and MidiMACS Separators(Miltenyi Biotec., Auburn, CA) was used according to the instruction manual. The purity of sorted cell populations was verified by flow cytometry.
4
Isolation of CD14+ Monocytes from Apheresis Blood
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Peripheral blood mononuclear cells (PBMNCs) were isolated from platelet apheresis blood waste (NHSBT, Bristol, UK) from anonymous healthy donors. PBMNC separation was performed using PBMNC Spin Medium (pluriSelect Life Science) as previously described.[58 , 59 ] Briefly, blood from apheresis cones was diluted 1:1 with Hank's Balanced Salt Solution (HBSS, Sigma) containing 0.6% acid citrate dextrose and layered over PBMNC spin medium. Samples were centrifuged to generate a density gradient and PBMNCs collected from the interface. CD14+ cells were isolated from PBMNCs using a magnetic micro‐bead CD14+ kit (Miltenyi Biotec), LS columns (Miltenyi Biotec), and MidiMACS separators (Miltenyi Biotec) as previously described.[60 ] CD14+ cells were stored frozen in 50% fetal bovine serum (FBS, Gibco) and 40% PBS with 10% DMSO (Sigma) in liquid nitrogen until required.
5
Isolation of CD4+ TEM Lymphocytes
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PBMCs were isolated from a healthy donor’s blood and cultured as explained earlier. Dead Cell Removal Microbead Kit (Miltenyi Biotec B.V & CO. KG) was used to eliminate the dead cells, and CD4+ TEM lymphocytes were isolated through magnetic cell sorting (negative selection) with the CD4+ Effector Memory T Cell Isolation Kit (Miltenyi Biotec B.V & CO. KG). Briefly, all types of cells except CD4+ TEM lymphocytes were labeled with a cocktail of monoclonal antibodies (biotin-conjugated anti-CD8, -CD14, -CD15, -CD16, -CD19, -CD34, -CD36, -CD45RA, -CD56, -CD123, -CD235a, -TCR γ/δ, and APC-conjugated anti-CCR7). Next, cells were incubated with anti-APC and anti-biotin secondary antibodies, both coupled with magnetic microbeads. The cell preparation was passed through LD Column (Miltenyi Biotec B.V & CO. KG) mounted on MidiMACS Separator (Miltenyi Biotec B.V & CO. KG), and untouched CD4+ TEM lymphocytes were collected as flow through.
6
Isolation and Culture of Corneal Endothelial Cells
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After 7 to 10 days, when the primary mixed culture showed cell islands with EC-like morphology, magnetic-activated cell sorting (MACS) was used to isolate CECs. For this purpose, the anti-human CD31 MicroBead Kit and LS columns on a MidiMACS separator (all from Miltenyi Biotec B.V.) were used according to the manufacturer's instructions. A TE concentration of 0.01% was used, and cells were maintained in DMEM þ 10% FCSi. CECs were resuspended and seeded on fibronectincoated 6-well plates in EGM-2MV medium (3 to 5 wells per eye, 1 ml fibronectin dilution per well, 1.5 ml EGM-2MV per well). Four to 7 days later, MACS was repeated to obtain sufficient pure CEC cultures, after which cells were seeded on 12-well plates (12 wells per eye, 0.5 ml fibronectin dilution per well, 1 ml EGM-2MV per well). All treatment experiments were performed during the second passage (i.e., after two MACS procedures) at near confluency and TriPure (Roche, Basel, Switzerland) was added directly afterward for RNA isolation.
7
Isolation and Transduction of Murine Sca-1+ HSPCs
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Sca-1+ isolation from murine bone marrow was performed by immunomagnetic bead selection as previously described.80 (link) Briefly, bone marrow from CD45.1+ donor mice was harvested and incubated with a biotinylated anti-Sca-1 antibody (Biolegend, San Diego, CA, USA) followed by incubation with anti-biotin microbeads (Miltenyi, Gaithersburg, MD, USA). Labeled cells were then passed through an LS column loaded on a MidiMACS Separator (Miltenyi). Sca-1+ cells were harvested and cultured in serum-free StemSpan medium (Stem Cell Technologies, Vancouver, BC, Canada) for 3 days in the presence of mouse SCF (100 ng/mL), mouse IL-3 (20 ng/mL), human IL-11 (100 ng/mL), and human Flt-3 ligand (100 ng/mL). All cytokines were purchased from R&D Systems (Minneapolis, MN, USA). Sca-1+ cells were transduced on day −1 and day 0 with half volume CD68-ECO-ET3-LV (LV production described in detail by Lytle et al.8 (link)) at a density of 2.0 × 106 Sca-1+ cells/mL and multiplicity of infection (MOI) of 13–63. Transduced CD45.1+ Sca-1+ HSPCs were harvested, washed, and resuspended in PBS and then transplanted by retro-orbital injection into preconditioned HA mice.
8
Isolation and Culture of Cell Lines and NK Cells
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The cell lines A549, CT26, and HEK293 were purchased from the American Type Culture Collection. The A549-GL cell line was generated using a lentiviral vector encoding for GFP and luciferase. CT26-hCAR and CT26-MICAmut were produced by infecting with lentiviral vectors encoding for the human Coxsackie and Adenovirus Receptor or the MICA*01mut1D gene [7 (link)], respectively. Cell lines were cultured in recommended culture media containing 10% fetal bovine serum (FBS) and antibiotics at 37ºC, 5% CO2.
PBMCs from healthy donors were isolated from blood by ficoll density gradient centrifugation and cultured in complete RPMI-1640 10% FBS. NK cells were isolated from total PBMCs using human CD56 microbeads, LS columns, and MidiMACS separator (Miltenyi Biotec), and cultured with NK MACs medium (Miltenyi Biotec) with 5% human AB serum (Biowest) and 200 IU/ml of IL2 (Clinigen).
PBMCs from healthy donors were isolated from blood by ficoll density gradient centrifugation and cultured in complete RPMI-1640 10% FBS. NK cells were isolated from total PBMCs using human CD56 microbeads, LS columns, and MidiMACS separator (Miltenyi Biotec), and cultured with NK MACs medium (Miltenyi Biotec) with 5% human AB serum (Biowest) and 200 IU/ml of IL2 (Clinigen).
9
Isolation and Cryopreservation of Tumor-Infiltrating Leukocytes
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Tumor tissue was obtained from tumors of patients who underwent surgery at the University of California, Los Angeles. All patients provided written informed consent. This study was conducted in accordance with the Declaration of Helsinki and under institutional review board approved protocol. Peripheral blood was drawn from patients prior to surgical resection of their tumors. Tumor tissue not needed for diagnosis was digested using the Miltenyi Brain Tumor Dissociation kit (Miltenyi Biotec, cat. 130-095-42) and gentleMACS dissociator (Miltenyi Biotec, cat. 130-093-235) and labeled with CD45+ microbeads (Miltenyi Biotec, cat. 130-045-801). CD45+ cells were positively selected for with Miltenyi LS columns (Miltenyi Biotec, cat. 130-042-401) and MidiMACS separator (Miltenyi Biotec, cat. 130-042-302). Collected CD45+ cells were then placed in Bambanker (Fisher Scientific, cat. 302-14681) and stored in liquid nitrogen. Peripheral blood mononuclear cells were collected in CPT tubes (BD Biosciences, cat: 362753), isolated according to the manufacturer's protocol, placed in freezing media made of 90% human AB serum (Fisher Scientific, cat. MT35060CI) and 10% dimethyl sulfoxide (Sigma, cat. C6295-50ML) and stored in liquid nitrogen.
10
Adoptive Transfer of 2D2 T Cells
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