The protocol for culture of EPCs was approved by the Institutional Review Board of Mackay Medical College, New Taipei City, Taiwan (reference number P1000002), and all subjects gave informed written consent before enrollment in this study. Peripheral blood (80 ml) was collected from healthy donors, and mononuclear cells were isolated by centrifugation on Ficoll-Paque PLUS (Amersham Biosciences, Uppsala, Sweden) according to the manufacturer's instructions. CD34-positive progenitor cells were isolated from the mononuclear cell fraction using a CD34 MicroBead kit and MACS Cell Separation System (Miltenyi Biotec, Bergisch Gladbach, Germany). CD34-positive EPCs were maintained and characterized as described previously [43 (link)]. Briefly, human CD34-positive EPCs were cultured in MV2 complete medium consisting of MV2 basal medium and growth supplement (PromoCell, Heidelberg, Germany), supplemented with 20% defined FBS (HyClone, Logan, UT). The cultures were seeded on 1% gelatin-coated plasticware and maintained at 37°C in a humidified 5% CO2 atmosphere.
Macs cell separation system
Manufactured by Miltenyi Biotec
Sourced in Germany, United States
The MACS cell separation system is a laboratory equipment designed for the magnetic separation of cells. It utilizes MACS microbeads, which are super-paramagnetic particles coated with antibodies, to selectively label target cells. The labeled cells can then be separated from the rest of the cell population using a MACS column and a magnetic field. This technology allows for the efficient isolation and purification of specific cell types from heterogeneous samples.
Automatically generated - may contain errors
Lab products found in correlation
Cd34 microbead kit, by Miltenyi Biotec (8 mentions)
Growth supplement, by PromoCell (4 mentions)
Ficoll paque plus, by Cytiva (4 mentions)
Mv2 basal medium, by PromoCell (4 mentions)
Anti p erk, by Cell Signaling Technology (2 mentions)
Trametinib, by Miltenyi Biotec (2 mentions)
Trametnib, by Selleck Chemicals (2 mentions)
Celltiter glo luminescentviability kit, by Promega (2 mentions)
Wallace plate reader, by Thermo Fisher Scientific (2 mentions)
Goat anti mouseig h l fitc, by Southern Biotech (2 mentions)
Cd5 microbeads, by Miltenyi Biotec (2 mentions)
Dnase 1, by Roche (2 mentions)
Collagenase, by Fujifilm (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Hyaluronidase, by Merck Group (2 mentions)
Dnase, by Merck Group (2 mentions)
Cd14 microbead, by Miltenyi Biotec (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Collagenase, by Merck Group (2 mentions)
Antibiotic antimycotic solution, by Thermo Fisher Scientific (2 mentions)
64 protocols using macs cell separation system
1
Isolation and Characterization of EPCs
2
Evaluating MEK Inhibitor Efficacy on Tumor Cells
3
Adoptive Transfer of Antigen-Specific CD8+ T Cells
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After final immunization, CD8+ T cells were purified from single-cell splenocyte suspensions from either GAS- or GAS/MAGE-A3-immunized HLA-A2 transgenic mice, with positive selection using a MACS cell separation system (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Subsequently, 1 × 107 purified CD8+ T cells (> 95% purity) were injected intravenously into nude mice 10 d after the mice were subcutaneously implanted with A549 cells.
4
Cytotoxic T-cell Activity in Tumor Microenvironment
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Cytotoxic T lymphocyte activity of tumor infiltrating lymphocytes was determined by standard 51Cr release assays. In brief, Neuro2a cells (target) were incubated with 0.2 mCi Na[51 ]CrO4 for 45 min at 37°C. Cells were washed twice with complete medium and transferred to round-bottom 96-well plates at 5 × 103 cells/well. CD8+ T-cells (effector) were purified from the unvaccinated growing and vaccinated shrinking total tumor digest using the MACS cell separation system (Miltenyi Biotec, Auburn, CA). Due to low abundance of the infiltrating lymphocytes in the growing tumors, purified T-cells were pooled together from four unvaccinated growing tumors for the assay. Effector T-cells were added to target tumor cells at varying numbers in a final volume of 0.2 ml to give the effector:target ratios as indicated in the Figure legends. After 4 hours incubation at 37°C, 0.1 ml of supernatant was harvested, and released radiolabel was determined by scintillation counting. Maximal release from targets was determined by treatment of cells with 1% Triton X-100, spontaneous release was determined from cultures of labeled targets incubated with medium only, and the formula used for determination of specific lysis was: [(experimental release − spontaneous release)/(maximal release − spontaneous release)] × 100.
5
DC and CD4+ T Cell Isolation
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For DC isolation, MLNs and PPs obtained from BALB/c mice were digested in 10% FCS-RPMI containing 0.5 mg/mL collagenase (FUJIFILM Wako Pure Chemical Corporation) with 10 µg/mL DNase I
(Roche Diagnostics GmbH, Mannheim, Germany), and a cell suspension was obtained by filtering the digestion. From the obtained whole cells, CD11c+ cells were magnetically purified
with a MACS cell separation system (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. CD11c+ purification with MACS was conducted twice to
achieve high purity, and CD11c+ cells were used as DCs. For CD4+ T cell isolation, splenocytes were obtained from DO11.10 mice. CD4+ cells were separated
from whole splenocytes with the MACS system. Cells were isolated from multiple mice and pooled to obtain the required number of cells.
(Roche Diagnostics GmbH, Mannheim, Germany), and a cell suspension was obtained by filtering the digestion. From the obtained whole cells, CD11c+ cells were magnetically purified
with a MACS cell separation system (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. CD11c+ purification with MACS was conducted twice to
achieve high purity, and CD11c+ cells were used as DCs. For CD4+ T cell isolation, splenocytes were obtained from DO11.10 mice. CD4+ cells were separated
from whole splenocytes with the MACS system. Cells were isolated from multiple mice and pooled to obtain the required number of cells.
6
Isolation and Characterization of Eosinophils
7
Bone Marrow Hematopoietic Progenitor Assay
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Bone marrow was isolated from mice 8 weeks post pIpC injection, and subjected to lineage depletion using the MACS cell separation system (Miltenyi Biotec). 10,000 lineage negative bone marrow cells were plated on Methocult M3434 (Stemcell Technologies), and analyzed at day 9-11. Colonies were disaggregated, and 200 cells were re-plated and scored for subsequent secondary and tertiary colony formation. Three mice were assayed per genotype, and experiments were performed in triplicate.
8
Isolation of Dermal Dendritic Cells
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LCs that migrated from skin explants were isolated as previously described (Nagao et al., 2009 (link)). In short, mouse ears were divided into dorsal and ventral halves, cartilage was removed, and the ear halves were then floated on 40 ml of complete RPMI 1640 medium supplemented with Amphotericin B at 37 °C in 10-cm petri dishes. The cells that migrated into the culture medium were collected after 24 h and were stored at 4 °C. These cells were pooled with cells that were harvested after an additional 24 h of skin explant incubation in newly replaced culture medium. EpCAM− CD11b+ dermal DCs were isolated from dermal cell suspensions using a MACS cell separation system (Miltenyi Biotec).
9
Isolation of Human Placental Macrophages
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Human placental macrophages (PMs) and fetal membrane tissues were isolated from placental tissue samples from women who delivered healthy infants at full term by cesarean section (without labor). Deidentified tissue samples were provided by the Cooperative Human Tissue Network, which is funded by the National Cancer Institute. All tissues were collected in accordance with the guidelines of the Vanderbilt University Institutional Review Board (approval 131607). Macrophage isolation occurred as previously described (89 (link)); briefly, placental villous tissue samples were minced followed by digestion with DNase, collagenase, and hyaluronidase (all from Sigma-Aldrich, St. Louis, MO). Cells were filtered and centrifuged, and CD14+ cells were isolated using the magnetic MACS Cell Separation system with CD14 microbeads (Miltenyi Biotec, Auburn, CA). Cells were incubated in RPMI 1640 medium (ThermoFisher, Waltham, MA) with 10% charcoal stripped fetal bovine serum (ThermoFisher) and 1% antibiotic/antimycotic solution (ThermoFisher) overnight at 37°C in 5% carbon dioxide. The following day, PMs were suspended in RPMI 1640 medium without antibiotic/antimycotic and distributed into polystyrene plates. Cells were incubated for at least 1 h prior to infection to allow for cell adherence to the plate or to poly-L-lysine-coated glass coverslips (Corning, Bedford, MA) for microscopy assays.
10
Isolation of CD34+ Progenitor Cells
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Peripheral
blood (80 mL) was collected from healthy volunteers with informed
consent before collection. The peripheral blood mononuclear cells
(PBMCs) were fractionated from other blood components by centrifugation
on Ficoll-Paque Plus (Amersham Biosciences, Uppsala, Sweden) based
on the manufacturer’s instructions. Utilizing CD34 MicroBead
kit and MACS Cell Separation System (Miltenyi Biotec, Bergisch Gladbach,
Germany), the CD34-positive progenitor cells were obtained from the
separated PBMCs. The isolation and maintenance of CD34-positive EPCs
were carried out as previously described.37 (link)
blood (80 mL) was collected from healthy volunteers with informed
consent before collection. The peripheral blood mononuclear cells
(PBMCs) were fractionated from other blood components by centrifugation
on Ficoll-Paque Plus (Amersham Biosciences, Uppsala, Sweden) based
on the manufacturer’s instructions. Utilizing CD34 MicroBead
kit and MACS Cell Separation System (Miltenyi Biotec, Bergisch Gladbach,
Germany), the CD34-positive progenitor cells were obtained from the
separated PBMCs. The isolation and maintenance of CD34-positive EPCs
were carried out as previously described.37 (link)
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