Microglia medium

Manufactured by ScienCell

Sourced in United States

Microglia medium is a specialized cell culture medium formulated to support the growth and maintenance of primary microglia cells. It provides the necessary nutrients and growth factors required for the optimal in vitro culture of these cells.

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Lab products found in correlation

Cd11b microbead, by Miltenyi Biotec (2 mentions) Cd11b c microbeads, by Miltenyi Biotec (2 mentions) Neuronal medium, by ScienCell (1 mentions) Poly l lysine (pll), by ScienCell (1 mentions) Neural tissue dissociation kit, by Miltenyi Biotec (1 mentions) Rpmi 1640 glutamax, by Thermo Fisher Scientific (1 mentions) Recombinant human il 34, by R&D Systems (1 mentions) Macs buffer, by Miltenyi Biotec (1 mentions) Ls column, by Miltenyi Biotec (1 mentions) Gm csf, by R&D Systems (1 mentions) Adult brain dissociation kit, by Miltenyi Biotec (1 mentions)

8 protocols using microglia medium

Isolation and Culture of Murine Microglia

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Brains resected from healthy, 5–8 week old C57BL/6 mice, were chopped and incubated with 1 mg/ml Collagenase IV, 2 mg/ml Dispase II (neutral protease), and 0.02 U/ml DNase I for 50 min at 37 °C. Red blood cells (RBC) were lysed with RBC lysis solution followed by a Percoll gradient for myelin separation. The resultant cell-suspension was then incubated with CD11b microbeads for microglia or with CD45 microbeads for total leukocytes, and the desired population was isolated on MACS MS magnetic columns. Murine microglia were then seeded on poly-L-Lysine (PLL)-coated plates in microglia medium (ScienCell, California, USA).

Yeini E., Ofek P., Pozzi S., Albeck N., Ben-Shushan D., Tiram G., Golan S., Kleiner R., Sheinin R., Israeli Dangoor S., Reich-Zeliger S., Grossman R., Ram Z., Brem H., Hyde T.M., Magod P., Friedmann-Morvinski D., Madi A, & Satchi-Fainaro R. (2021). P-selectin axis plays a key role in microglia immunophenotype and glioblastoma progression. Nature Communications, 12, 1912.

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Isolation and Culture of Primary Neural Cells

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Primary neurons, microglia, and astrocytes isolated from CD1 mice were obtained from ScienCell Research Laboratories (Carlsbad, CA). Neurons and microglia were seeded onto 24 well cell culture plates coated with Poly-L-Lysine (10 μg/ml) (ScienCell Research Laboratories), while astrocytes were first expanded in a Poly-L-Lysine coated T-75 flask prior to seeding onto Poly-L-Lysine coated 24 well cell culture plates. Neurons, microglia and astrocytes were maintained in neuronal medium, microglia medium, and animal astrocyte medium (ScienCell Research Laboratories) respectively, until confluent and ready to use.

Michael B.D., Bricio-Moreno L., Sorensen E.W., Miyabe Y., Lian J., Solomon T., Kurt-Jones E.A, & Luster A.D. (2020). Astrocyte- and Neuron-Derived CXCL1 Drives Neutrophil Transmigration and Blood-Brain Barrier Permeability in Viral Encephalitis. Cell reports, 32(11), 108150.

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Isolation of Primary Microglia from Human Tissue

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Human tissue samples were dissociated using a Neural Tissue Dissociation Kit (Miltenyi Biotec, Bergisch-Gladbach, Germany), according to the manufacturer’s instructions (Figure 1). The cell suspension was incubated with CD11b microbeads (Miltenyi Biotec) in the MACS buffer (Miltenyi Biotec) for 15 min at 4°C. Afterward, the cells were washed, resuspended, and transferred to an LS column (Miltenyi Biotec) within a magnetic field. The positively selected (CD11b⁺) microglia were collected and resuspended in the Microglia Medium (ScienCell, Carlsbad, CA, USA). Primary microglia (pMG) were plated on culture dishes at a density of 3 × 10⁵ cells/mL and cultured overnight in standard culture conditions (37 °C, 5% CO₂). After overnight incubation, culture supernatant and non-adherent cells were removed. Microglia were cultured in RPMI-1640 Glutamax (Invitrogen, Carlsbad, CA, USA) supplemented with 1% antibiotic/antimycotic, recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF) (10 ng/mL; R&D Systems, Minneapolis, MN, USA) and recombinant human IL-34 (100 ng/mL; R&D Systems) for 5 days.

Tanaka S., Ohgidani M., Hata N., Inamine S., Sagata N., Shirouzu N., Mukae N., Suzuki S.O., Hamasaki H., Hatae R., Sangatsuda Y., Fujioka Y., Takigawa K., Funakoshi Y., Iwaki T., Hosoi M., Iihara K., Mizoguchi M, & Kato T.A. (2021). CD206 Expression in Induced Microglia-Like Cells From Peripheral Blood as a Surrogate Biomarker for the Specific Immune Microenvironment of Neurosurgical Diseases Including Glioma. Frontiers in Immunology, 12, 670131.

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Microglia Enrichment from Brain Cells

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The cell pellet consisting of a mixture of all brain cells was further subjected to magnetic cells sorting for microglia enrichment using CD11b/c microbeads (Miltenyi Biotec), according to manufacturer protocol. The retrieved cells of the non-enriched and enriched fractions were suspended in microglia medium (ScienCell Research Labs. Inc.), and kept overnight until processing for flow cytometric analysis the next day.

Toledano Furman N., Gottlieb A., Prabhakara K.S., Bedi S., Caplan H.W., Ruppert K.A., Srivastava A.K., Olson S.D, & Cox CS J.r. (2020). High-resolution and differential analysis of rat microglial markers in traumatic brain injury: conventional flow cytometric and bioinformatics analysis. Scientific Reports, 10, 11991.

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Investigating Microglia Cell Responses

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Rat microglial (RM) cells were purchased from ScienCell™ (#R1900, ScienCell™ Research Laboratories, USA), which had been isolated from the cerebral cortex of two-day-old rat pups. Cells were delivered frozen as a secondary culture. When initiating the subculture from the cryopreserved cells, the cells were cultured in an incubator at 5% CO₂ and 37°C, using microglia medium (#1901, ScienCell™ Research Laboratories). Then the cells were seeded on coverslips in 12-well plates at a concentration of 5 × 10⁶ cells per well that contained phenol red-free DMEM (HyClone, USA). The RM cells were treated with 10 ng/mL lipopolysaccharide (LPS) (#L6529, Sigma-Aldrich) for 24 h. This in vitro study included the following groups: control RM cell group (RM), LPS-treated RM cell group (LPS), LPS + U50488H RM cell group (LU), LPS + Ac-YVAD-cmk (SML0429, Sigma-Aldrich) RM cell group (LAC), and LPS + U50488H + RS09 (#5928, TOCRIS, UK) RM cell group (LUR). The cells were treated with 100 ng/mL LPS for 24 h, followed by exposure to 1 μmol/L of U50448H for 30 min. Ac-YVAD-cmk was dissolved in 500 μl of DMSO, and the working concentration was 50 μM (27 μg/ml), which was added to the cells for 30 min before the LPS stimulation.

Song P., Yi Z., Fu Y., Song D., Chen K., Zheng J., Sun Y, & Diao Y. (2021). Reversing Postcardiopulmonary Bypass Associated Cognitive Dysfunction Using k-Opioid Receptor Agonists to Regulate Microglial Polarization via the NLRP3/Caspase-1 Pathway. Journal of Healthcare Engineering, 2021, 3048383.

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Culturing Primary Neonatal Mouse Microglia

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Primary neonatal mouse microglia (Sciencell, Cat#M1900–57) were cultured in poly-lysine coated 96-well plates with Microglia Medium (Sciencell, Cat #1901). Cultures were kept in an incubator at 37 °C, 5% CO₂. Media was refreshed 24 h after plating. 4 days after plating, media was removed and replaced with fresh media containing a stimulus.

Cho C.E., Damle S.S., Wancewicz E.V., Mukhopadhyay S., Hart C.E., Mazur C., Swayze E.E, & Kamme F. (2019). A modular analysis of microglia gene expression, insights into the aged phenotype. BMC Genomics, 20, 164.

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Microglia Isolation and CSF Exposure

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The brain tissue was dissociated using the Adult Brain Dissociation Kit (Miltenyi Biotec) according to the manufacturer’s protocol. Microglia were isolated using the CD11b MicroBeads (Miltenyi Biotec) and then cultured (37 °C, 5% CO₂) in Microglia Medium (ScienCell) with 5% fetal bovine serum,1% penicillin, 1% streptomycin and Microglia growth supplement for 7 days. At day 8, cultures were exposed to each individual CSF sample for 24 h. The exposure medium contained 25% CSF or PBS in Microglia Medium.

Zhang X., Wang R., Chen H., Jin C., Jin Z., Lu J., Xu L., Lu Y., Zhang J, & Shi L. (2022). Aged microglia promote peripheral T cell infiltration by reprogramming the microenvironment of neurogenic niches. Immunity & Ageing : I & A, 19, 34.

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Microglia Enrichment from Brain Cells

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Gottlieb A., Toledano-Furman N., Prabhakara K.S., Kumar A., Caplan H.W., Bedi S., Cox CS J.r, & Olson S.D. (2022). Time dependent analysis of rat microglial surface markers in traumatic brain injury reveals dynamics of distinct cell subpopulations. Scientific Reports, 12, 6289.

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