Splenic iNKT cells from MyD88 control or cKO animals were enriched using magnetic depletion on an AutoMACS or OctoMACS (Miltenyi) with CD19 and CD8 MicroBeads (Miltenyi). Samples were then i.v. injected into B6.SJL recipients and 3 hours post-injection recipients were infected with MCMV. At 36 hours post-infection, recipients were sacrificed and splenic iNKT cells were enriched using magnetic depletion on an AutoMACS or OctoMACS (Miltenyi) with CD19 and CD8 MicroBeads (Miltenyi). The entire splenic negative fraction and liver samples were then stained to determine IFN-γ production.
Octomacs
Manufactured by Miltenyi Biotec
Sourced in Germany
The OctoMACS is a magnetic cell separation system designed for precise and efficient isolation of target cells from heterogeneous cell populations. It utilizes magnetic beads coated with specific antibodies to selectively bind and separate the desired cell types.
Automatically generated - may contain errors
Lab products found in correlation
Automacs, by Miltenyi Biotec (3 mentions)
Cd19 microbeads, by Miltenyi Biotec (2 mentions)
Cd8α microbeads, by Miltenyi Biotec (2 mentions)
Cd19 and cd8 microbeads, by Miltenyi Biotec (1 mentions)
Cd45 magnetic beads, by Miltenyi Biotec (1 mentions)
Ficoll paque premium, by GE Healthcare (1 mentions)
Dnase, by Merck Group (1 mentions)
Collagenase, by Merck Group (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Ripa buffer, by Cell Biolabs (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Hyaluronidase, by Merck Group (1 mentions)
Liberase, by Roche (1 mentions)
Collagenase xi, by Merck Group (1 mentions)
Macs multistand, by Miltenyi Biotec (1 mentions)
Cd11b microglia microbeads, by Miltenyi Biotec (1 mentions)
Myelin removal beads 2, by Miltenyi Biotec (1 mentions)
Quadromacs, by Miltenyi Biotec (1 mentions)
Gentlemacs octo dissociator with heater, by Miltenyi Biotec (1 mentions)
Neural tissue dissociation kit, by Miltenyi Biotec (1 mentions)
10 protocols using octomacs
1
MCMV Infection Assay using iNKT Cells
2
Adoptive Transfer of CD8+ T Memory Cells
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Samples were enriched for CD8+ cells using an AutoMACS or OctoMACS and either CD8α MicroBeads or CD19 MicroBeads (Miltenyi Biotech). 50,000 CD8+ TM cells (CD8+KLRG1−CD27+) sorted from long-term infected KbDb−/− spleens or KbDb−/−Qa-1−/− spleens were adoptively transferred per recipient. For survival studies, cells were injected i.v. into RAG1/KbDb−/− mice and three hours post-injection, recipients were infected with MCMV. For secondary MCMV infections, cells were injected i.v. into KbDb−/−.SJL mice and recipients were infected two or three hours post-injection.
3
Isolation of Lung Organoid Cells
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BAL fluid was collected from uninjured control or injured animals as described and red blood cells were lysed (0.154M NH4Cl/10mM KHCO3/0.127mM EDTA). The remaining cell suspension was incubated with CD45 magnetic beads (Miltenyi #130-052-301) and positively selected using the OctoMACS (Miltenyi) platform. Cells were then passed through a 0.4μm cell strainer prior to lung organoid co-culture.
4
Enriching and Characterizing CAR NK Cells
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Three to four days after transduction, CAR NK cells were enriched on an OctoMACS™ Separator using MS columns or on a QuadroMACS™ Separator using LS columns after staining with CD34 microbeads (clone QBend-10) according to the manufacturer’s instructions (Miltenyi Biotec) and similarly as described previously for CAR T cells (28 (link)). In order to investigate the MACS enrichment efficiency, the CAR NK cell purity and yield, and the expression of the different CAR constructs on the transduced NK cells, the three cell fractions—before MACS, flowthrough, and after MACS—were collected and analyzed by flow cytometry for BFP expression and/or CD34 positivity. Before any functional analyses were performed, the MACS-enriched CAR NK cells were further cultured for at least an additional 2 days in a complete NK MACS medium.
5
Ferret Lung Immune Cell Isolation
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Whole lungs were removed from each ferret. The lungs were dissected into small pieces and placed into a 12.5 ml solution of collagenase (715 collagenase units/ml) (Sigma-Aldrich) and DNase (350 DNase units/ml) (Sigma-Aldrich). Lungs were placed into gentleMACS C-tubes and agitated whilst incubating, 37 oC for 1 h on an OctoMACS (Miltenyi Biotec, Surrey, UK). Partially digested lung tissue was then dissociated using an OctoMACS. The tissue solution was passed through two cell sieves (100 µm then 70 µm) and then layered with Ficoll®- Paque Premium (GE Healthcare, Hatfield, UK). Density gradient centrifugation was carried out at 400g for 30 min. Buffy coats containing lymphocytes were collected and washed with medium by pelleting cells via centrifugation at 400g for 10 min. The cells were counted using a Via1- cassette and a Nucleocounter-200 before cryopreservation in 95% FCS/5% v/v DMSO. Cryopreserved cells were then frozen at −80 °C in controlled rate freezer containers overnight, before transfer to liquid nitrogen (vapour phase).
6
Tissue Homogenization and Protein Extraction
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70–150mg of tumor pieces were chopped into small pieces in RIPA buffer containing proteinase inhibitor (Cell BioLabs) and homogenized using OctoMACS (Miltenyi Biotec). The homogenate was centrifuged at 400g for 5 minutes followed by further centrifugation at 10,000g for 10 minutes at 4C°. The supernatant was collected and stored at −80C°. Protein concentration was measured using DC protein assay kit (BioRad). The sample concentrations were normalized prior to the submission for the analysis by Eve Technologies DM-44 mouse Discovery Assay panel.
7
Adoptive Transfer of CD8+ T Memory Cells
8
Lung and Lymph Node Single-Cell Isolation
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Lungs were chopped with a razor blade or underwent automated digestion in C-Tubes using a heated OctoMACS (Miltenyi Biotech, Bergisch Gladbach, North Rhine-Westphalia, Germany), digested with 250 μg/ml Collagenase XI (C7657; Sigma, St. Louis, MO, USA), 50 μg/ml Liberase (145495; Roche, Basel, Switzerland), 1 mg/ml Hyaluronidase (h3506; Sigma, St. Louis, MO, USA), and 200 μg/ml DNase I (DN25; Sigma, St. Louis, MO, USA) in RPMI 1640 at 37°C for 30 minutes. Tissues were vortexed and pipetted vigorously every 15 minutes until fully digested and filtered through an 80-micron mesh, washed, lysed for 2 minutes with ammonium-chloride-potassium (ACK) buffer to reduce red blood cells, washed with PBS, and resuspended in 2% fetal bovine serum in PBS. Single-cell suspensions of lymph nodes were prepared by mechanical dissociation. All tissues were filtered immediately prior to data collection.
9
Microglia Isolation from Mouse Brain
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Neural Tissue Dissociation Kit (P), Myelin Removal Beads II, and CD11b (microglia) MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) were used for magnetic isolation of microglial cells from adult APPPS1/Cx3cr1‐eGFP−/+, APPPS1, APPPS1/Nlrp3−/−/Cx3cr1‐eGFP/+, and APPPS1/Nlrp3−/− mice brains, in accordance with the manufacturer's guidelines. Briefly, brains were dissected after perfusion with PBS, enzymatically digested using the Neural Tissue Dissociation Kit. Cells were incubated for 15 min at 4°C with Myelin Removal Beads II (Miltenyi Biotec, Bergisch Gladbach, Germany) and separated from myelin in a magnetic field using LS columns, MACS MultiStand, and QuadroMACS (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were incubated with CD11b (microglia) MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) for 15 min followed by separation of CD11b‐positive cells in a magnetic field using MS columns and OctoMACS (Miltenyi Biotec, Bergisch Gladbach, Germany).
10
Isolation of Neonatal Astrocytes from Mice
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Neonatal astrocytes were isolated by using MACS from p 4-8 C57BL/6J mice. Cerebral tissue was isolated, meninges removed and brains of two mice pooled in a C-tube for tissue dissociation with the Neural Tissue Dissociation Kit (Miltenyi, 130-092-628) on program 37C_NTDK_1 of the gentleMACS™ Octo Dissociator with Heaters (Miltenyi Biotec, 130-096-427) allowing for simultaneous enzymatic and mechanical tissue disruption. Afterwards, the cell suspension was washed with Hanks' Balanced Salt solution without calcium and magnesium (HBSS, Thermo Fisher, 14170138) and astrocytes magnetically labelled with ACSA-2 microbeads (Miltenyi Biotec, 130-097-678), according to manufacturer's protocol. The resulting cell suspension was ltered once through a 70 µm pre-separation lter, consecutively passed through two MS columns (Miltenyi Biotec, 130-042-201) placed on an OctoMACS™ manual separator and ushed into Eppendorf tubes in 0.5 % BSA in PBS, pH 7.2 or cell culture medium (see section on astrocyte culture below). Cell pellets were collected, snap-frozen in liquid nitrogen and stored at -80 °C until further use.
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