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Automacs pro separation system

Manufactured by Miltenyi Biotec
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Sourced in Germany

The AutoMACS Pro Separation System is a fully automated magnetic cell separation device. It is designed to perform rapid, high-purity cell separations with minimal hands-on time. The system utilizes magnetic beads coated with antibodies to selectively isolate target cell populations from complex samples.

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Lab products found in correlation

Buffy coat, by GE Healthcare (2 mentions) Cd14 microbead conjugated antibody, by Miltenyi Biotec (2 mentions) Mt35060ci, by Thermo Fisher Scientific (2 mentions) L alanine l glutamine, by Thermo Fisher Scientific (2 mentions) Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions) Ficoll hypaque, by GE Healthcare (2 mentions) Probenecid, by Merck Group (1 mentions) Liveblazer fret b g loading kit, by Thermo Fisher Scientific (1 mentions) Anti cd4 microbeads, by Miltenyi Biotec (1 mentions) Rna seq preparation kit, by Illumina (1 mentions) Ack lysing buffer, by Lonza (1 mentions) Moflo xdp, by Beckman Coulter (1 mentions) Anti apc microbeads, by Miltenyi Biotec (1 mentions) Facsaria 2, by BD (1 mentions) Facsaria, by BD (1 mentions)

7 protocols using automacs pro separation system

1

Yop Translocation Quantification in CD4+ T Cells

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For in vitro analysis of Yop translocation, total CD4+ T cells were isolated from spleens and LNs of Foxp3hCD2 mice using anti-CD4-Microbeads and the autoMACS Pro separation system (Miltenyi Biotec). Subsequently, cells were co-cultured with Yptb-WT-Bla or Yptb-ΔT3SS-Bla at the indicated MOIs at 37 °C for 1 h and washed twice with RPMI medium supplemented with 50 μg/mL gentamicin to eliminate bacteria. Cells were stained for cell surface markers anti-CD4 and anti-hCD2 and labeled with CCF4-AM, using the LiveBLAzer-FRET B/G loading kit (Thermo Fisher Scientific) for 1 h at 24 °C in the presence of 1.5 mM probenecid (Sigma-Aldrich) and 50 μg/mL gentamicin. Finally, cells were harvested and analyzed by flow cytometry.
Elfiky A., Bonifacius A., Pezoldt J., Pasztoi M., Chaoprasid P., Sadana P., El-Sherbeeny N., Hagras M., Scrima A., Dersch P, & Huehn J. (2018). Yersinia Pseudotuberculosis Modulates Regulatory T Cell Stability via Injection of Yersinia Outer Proteins in a Type III Secretion System-Dependent Manner. European Journal of Microbiology & Immunology, 8(4), 101-106.
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2

Isolation and Culture of Human Monocytes

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Human normal monocytes (CD14+ cells) were isolated by the AutoMACS Pro Separation System (Miltenyi Biotech, Auburn, CA) from the mononuclear cells fraction of normal peripheral blood. Briefly, buffy coat was purchased from Interstate Blood Bank (Memphis, TN) and the mononuclear cell layer was separated by Ficoll Hypaque (17144003, GE Lifesciences) density gradient separation. Mononuclear cells were treated with red blood cell lysis buffer to remove red blood cells and then incubated with CD14 microbead-conjugated antibody (130–050-201, Miltenyi Biotech, Auburn, CA) for 15 min at 4°C. CD14 positive cells were then isolated using the positive selection program according to the manufacturer’s protocol. One million CD14+ cells were plated in macrophage culture media, Iscove’s modified Dulbecco’s medium (IMDM) (12440053, Thermo fisher) supplemented with 10% human AB serum (MT35060CI, Fisher Scientific), 1% NEAA (11–140-050, Fisher), 2 μM L-alanine-L-glutamine (SH3003402, Fisher), per each well of a 6-well plate and cultured for 7 days. After incubation, most of the cells were adherent to the plastic surface and stained positive for CD14 and other markers specific for macrophages.
Deng M., Gui X., Kim J., Xie L., Chen W., Li Z., He L., Chen Y., Chen H., Luo W., Lu Z., Xie J., Churchill H., Xu Y., Zhou Z., Wu G., Yu C., John S., Hirayasu K., Nguyen N., Liu X., Huang F., Li L., Deng H., Tang H., Sadek A.H., Zhang L., Huang T., Zou Y., Chen B., Zhu H., Arase H., Xia N., Jiang Y., Collins R., You M.J., Homsi J., Unni N., Lewis C., Chen G.Q., Fu Y.X., Liao X.C., An Z., Zheng J., Zhang N, & Zhang C.C. (2018). LILRB4 signaling in leukemia cells mediates T cell suppression and tumor infiltration. Nature, 562(7728), 605-609.
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3

CD15+ Cerebellar Cell Isolation and RNA-Seq Analysis

Cited 1 time
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CD15+ cells were MACS enriched from P10 and P28
Utx+/+ or UtxF/FAtoh1-Cre SmoM2 cerebella. Dissociated cerebellar cells were
incubated with anti-mouse CD15 microbeads (Miltenyi), and CD15+ cells
were isolated by the AutoMACS Pro Separation System (Miltenyi Biotech) according
to the manufacturer’s protocol. The bulk RNA-seq used
Utx+/+ and
Utx+/Y tumor mice as wildtype and
UtxF/F tumor mice as mutants. Total RNAs
were extracted, and RNA-seq libraries were prepared using the Illumina RNA-Seq
Preparation Kit and sequenced on a HiSeq 2500 sequencer at the University of
Texas Southwestern Sequencing Core Facility. The expression levels of genes were
quantified by a Kallisto program (72 ),
and the differentially expressed genes were detected by an EdgeR program (73 (link)). Genes with a count-per-Million (CPM)
less than 1 in more than 2 samples were excluded. The differentially expressed
genes with a fold change larger than 2 and an FDR<0.05 were selected as
UTX-regulated genes. Gene ontology analysis was performed using DAVID tools
(http://david.abcc.ncifcrf.gov/).
Yi J., Shi X., Xuan Z, & Wu J. (2020). Histone Demethylase UTX/KDM6A Enhances Tumor Immune Cell Recruitment, Promotes Differentiation and Suppresses Medulloblastoma. Cancer letters, 499, 188-200.
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4

Allogeneic Bone Marrow Transplant in Mice

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C57BL/6 donor mice were euthanized by CO2 asphyxiation. Bone marrow (BM) cells were harvested from the tibias and fibulas using mortar and pestle and depleted of erythrocytes using ACK Lysing Buffer (Lonza, Walkersville, MD). CD3 microbeads were used on single-cell suspensions to deplete T cells using the AutoMACS Pro separation system (Miltenyi Biotec, Auburn, CA). Splenic T cells were isolated by negative selection. 4 + 4Gy irradiated Balb/c animals were injected intravenously with C57BL/6 T cell depleted BM (5X106 Cells) and T cells (2X106 Cells, 1X106 and 0.5X106) on the same day. BM alone group received only T cell depleted C57BL6 BM and not C57BL/6 splenic T cells. IFNγ licensed MSCs were infused intraperitoneally on days 2 and 8. In some situations, treatment was performed on days 1,3 and 5. Control group received only PBS. Mice were weighed individually biweekly, and the mean weight of each treatment group was calculated at each time point and was compared with the day 0 weight. Examination for moribund mice was performed by a veterinarian and veterinary technicians who were blinded to the experimental groups, and assessed the mice daily in accordance with approved institutional protocols.
Chinnadurai R., Bates P.D., Kunugi K.A., Nickel K.P., DeWerd L.A., Capitini C.M., Galipeau J, & Kimple R.J. (2021). Dichotomic Potency of IFNγ Licensed Allogeneic Mesenchymal Stromal Cells in Animal Models of Acute Radiation Syndrome and Graft Versus Host Disease. Frontiers in Immunology, 12, 708950.
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5

Isolation of CD4+CD8- Foxp3- Thymocytes

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CD4+CD8− single‐positive (SP) Foxp3− thymocytes were isolated from 4‐ to 6‐week‐old male Foxp3hCD2 reporter mice (BALB/c background). Single‐cell suspensions from thymi were labeled with anti‐hCD2‐FITC‐conjugated antibody, anti‐CD4‐PacificBlue‐conjugated antibody, anti‐CD8α‐APC‐conjugated antibody, and anti‐APC microbeads (Miltenyi Biotec). Using the autoMACS® Pro Separation System (Miltenyi Biotec), APC‐labeled CD8α+ cells were depleted. From the negative fraction, CD4SP Foxp3hCD2− thymocytes were sorted using a FACSAria™ II (BD Biosciences), a FACSAria™ (BD Biosciences), or a MoFlo XDP (Beckman Coulter).
Herppich S., Beckstette M, & Huehn J. (2021). The thymic microenvironment gradually modulates the phenotype of thymus‐homing peripheral conventional dendritic cells. Immunity, Inflammation and Disease, 10(2), 175-188.
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6

Isolation and Culture of Human Monocytes

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Human normal monocytes (CD14+ cells) were isolated by the AutoMACS Pro Separation System (Miltenyi Biotech, Auburn, CA) from the mononuclear cells fraction of normal peripheral blood. Briefly, buffy coat was purchased from Interstate Blood Bank (Memphis, TN) and the mononuclear cell layer was separated by Ficoll Hypaque (17144003, GE Lifesciences) density gradient separation. Mononuclear cells were treated with red blood cell lysis buffer to remove red blood cells and then incubated with CD14 microbead-conjugated antibody (130–050-201, Miltenyi Biotech, Auburn, CA) for 15 min at 4°C. CD14 positive cells were then isolated using the positive selection program according to the manufacturer’s protocol. One million CD14+ cells were plated in macrophage culture media, Iscove’s modified Dulbecco’s medium (IMDM) (12440053, Thermo fisher) supplemented with 10% human AB serum (MT35060CI, Fisher Scientific), 1% NEAA (11–140-050, Fisher), 2 μM L-alanine-L-glutamine (SH3003402, Fisher), per each well of a 6-well plate and cultured for 7 days. After incubation, most of the cells were adherent to the plastic surface and stained positive for CD14 and other markers specific for macrophages.
Deng M., Gui X., Kim J., Xie L., Chen W., Li Z., He L., Chen Y., Chen H., Luo W., Lu Z., Xie J., Churchill H., Xu Y., Zhou Z., Wu G., Yu C., John S., Hirayasu K., Nguyen N., Liu X., Huang F., Li L., Deng H., Tang H., Sadek A.H., Zhang L., Huang T., Zou Y., Chen B., Zhu H., Arase H., Xia N., Jiang Y., Collins R., You M.J., Homsi J., Unni N., Lewis C., Chen G.Q., Fu Y.X., Liao X.C., An Z., Zheng J., Zhang N, & Zhang C.C. (2018). LILRB4 signaling in leukemia cells mediates T cell suppression and tumor infiltration. Nature, 562(7728), 605-609.
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7

Monocyte Transfer to Colitic Mice

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Bone marrow was flushed from the tibias and femurs of donor Cx3cr1-gfp knock-in mice that had been challenged with DSS for 6 days to activate and expand monocytes. After lysing red blood cells with ACK lysing buffer, the cells were stained with phycoerythrin/Cy5-or Alexa-647-conjugated antibodies against CD3, CD19, NK1.1, Ter119, I-A/I-E, B220, and Ly6G. Lineage-marker-positive cells were depleted with anti-Cy5/Alexa-647 microbeads and an AutoMACSpro separation system (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturers' instructions. Cx3cr1 int , c-kit À , CD11c À , and SSC low monocytes were then sorted with a FACSAria III. The purified monocytes were transferred intravenously into the tail vein of WT hosts (CD45.1 À CD45.2 þ ) 4 days after the start of DSS treatment. After transferring, the host colons were analyzed at various time points.
Nakanishi Y., Sato T., Takahashi K, & Ohteki T. (2018). IFN-γ-dependent epigenetic regulation instructs colitogenic monocyte/macrophage lineage differentiation in vivo. Mucosal immunology, 11(3).
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