Adult microglia isolation was performed using MACS, as previously described [47 (link)]. Briefly, mice were anesthetized with avertin and transcardially thoroughly perfused with PBS to remove circulating blood cells in the CNS. Each dissected brain was chilled on ice and then minced in enzymatic digestion buffer containing 0.2% Collagenase Type 3 (Worthington, LS004182) and 3 U/mL Dispase (Worthington, LS02104). Minced brain tissue was then incubated at 37°C for 45 min. The enzymatic digestion was stopped with inactivation buffer containing 2.5 mM EDTA (Thermofisher, 15575020) and 1% fetal bovine serum (Invitrogen, 10082147). The digested brain tissue was then triturated in a serological pipette several times before passing through a 70-μm filter. The homogenate was then depleted of myelin using myelin removal beads (Miltenyi Biotec, 130-096-733) and magnetic LD column (Miltenyi Biotec, 130-042-901). The elute was enriched for microglia with CD11b magnetic beads (Miltenyi Biotec, 130-049-601) and MS column (Miltenyi Biotec, 130-042-201).
Magnetic ld column
Manufactured by Miltenyi Biotec
The Magnetic LD column is a lab equipment designed for the separation and enrichment of magnetically labeled cells. It utilizes a strong magnetic field to retain the labeled cells within the column, while allowing unlabeled cells to pass through. The core function of the Magnetic LD column is to facilitate the isolation and purification of specific cell populations from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Myelin removal beads, by Miltenyi Biotec (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (4 mentions)
Dispase, by Worthington (4 mentions)
Magnetic ms column, by Miltenyi Biotec (4 mentions)
Cd11b magnetic bead, by Miltenyi Biotec (3 mentions)
Collagenase type 3, by Worthington (2 mentions)
Ms column, by Miltenyi Biotec (1 mentions)
Dnase, by Merck Group (1 mentions)
Cd11b beads, by Miltenyi Biotec (1 mentions)
Cd11b microbead, by Miltenyi Biotec (1 mentions)
Ls004182, by Worthington (1 mentions)
Ls02104, by Worthington (1 mentions)
6 protocols using magnetic ld column
1
Isolation of Adult Mouse Microglia
2
Isolation of Adult Mouse Microglia
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Adult microglia were isolated by magnetic activated cell sorting as previously described23 . Briefly, mice were anesthetized with avertin and transcardially perfused with PBS to remove circulating blood cells in the central nervous system. Dissected brains were digested with 3% collagenase type 3 (Worthington, LS004182) and 3 U/ml dispase (Worthington, LS02104) and incubated at 37 °C for 45 min. Digestion was stopped with inactivation buffer containing 2.5mM EDTA (Thermofisher, 15575020) and 1% fetal bovine serum (Invitrogen, 10082147). Tissue was then triturated in a serological pipette several times and passed through a 70-μm filter. Myelin in the homogenate was depleted with myelin removal beads (Miltenyi Biotec, 130–096-733) and a magnetic LD column (Miltenyi Biotec, 130–042-901). Microglia were isolated from the elutant with CD11b magnetic beads (Miltenyi Biotec, 130–049-601) and a magnetic MS column (Miltenyi Biotec, 130–042-201).
3
Isolation of Adult Mouse Microglia
4
Murine Microglia Isolation and RNA-seq
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Adult microglia were isolated from 3-to 4-month-old mTrem2 +/+ , hTREM2 R47H/+ , and hTREM2 R47H/R47H mice as described 36 . Briefly, after perfusion, brains were chopped with a razor blade, incubated with 3% collagenase type 3 (Worthington), 3 U/ml dispase (Worthington) and DNase (Millipore Sigma) at 37°C, inactivated with 2.5 mM EDTA (ThermoFisher Scientific) and 1% fetal bovine serum (FBS) (Invitrogen), filtered through a 70-µm filter, centrifuged at 300 g for 5 min at 18°C and resuspended in fluorescence-activated cell sorting (FACS) buffer. Samples were incubated with myelin-removal beads (Miltenyi Biotec) for 15 min at 4°C, passed through a magnetic LD column (Miltenyi Biotec), centrifuged at 300 g for 10 min, and resuspended in FACS buffer. Cells were magnetically separated and sorted with CD11b beads (Miltenyi Biotec) and a magnetic MS column (Miltenyi Biotec). CD11b-positive cells were centrifuged at 300 g for 10 min, and RNA was extracted for RNA-seq.
5
Isolation of Adult Mouse Microglia
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Adult microglia were isolated using magnetic-activated cell sorting (MACS) as described before70 . Briefly, anesthetized mice were thoroughly transcardially perfused with cold PBS to remove circulating blood cells in the CNS. Dissected brains were chilled on ice and minced in digestion media containing 0.2% collagenase type 3 (LS004182, Worthington) and 3 U/mL dispase (LS02104, Worthington). After 37 °C incubation for 45 min, digestion was inactivated by 2.5 mM EDTA (15575020, Thermofisher) and 1% fetal bovine serum (10082147, Thermofisher). Digested brain tissues were triturated by serological pipette to the cell suspension and passed through a 70 μm cell strainer. Myelin in the cell suspension was depleted by myelin removal beads (130-096-733, Miltenyi Biotec) and magnetic LD columns (130-042-901, Miltenyi Biotec). Adult microglia were finally enriched from the eluant by CD11b MicroBeads (130-049-601, Miltenyi Biotec) and magnetic MS column (130-042-201, Miltenyi Biotec) for RNA isolation.
6
Isolation and Cryopreservation of PBMCs and NK Cells
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Fresh buffy coats were diluted in PBS and carefully layered onto Ficoll for PBMC isolation by density gradient centrifugation (400 g/40 min/RT, acceleration/deceleration at 0). The PBMC layer was harvested into 2 × 50 mL tubes and washed in 50 mL PBS each, centrifuged at 400 g/10 min/RT. The cell pellet was resuspended in fresh 50 mL PBS and centrifuged at 300 g/5 min/RT. The cell pellets were then pooled and washed in a final 50 mL PBS at 200 g/10 min/RT to yield the isolated PBMCs.
Cryopreserved PBMCs were quickly thawed in a 37°C water bath and transferred into 10 mL of cold RPMI‐1640 + 10% heat‐inactivated FCS. Cells were centrifuged at 300 g/5 min/RT and washed in a further 10 mL of media prior to isolating NK cells.
NK cells were isolated from fresh or cryopreserved PBMCs using the NK cell isolation kit, human (Miltenyi Biotec) and magnetic LD columns (Miltenyi Biotec) to yield the final purified PB‐derived NK cell starting material.
Cryopreserved PBMCs were quickly thawed in a 37°C water bath and transferred into 10 mL of cold RPMI‐1640 + 10% heat‐inactivated FCS. Cells were centrifuged at 300 g/5 min/RT and washed in a further 10 mL of media prior to isolating NK cells.
NK cells were isolated from fresh or cryopreserved PBMCs using the NK cell isolation kit, human (Miltenyi Biotec) and magnetic LD columns (Miltenyi Biotec) to yield the final purified PB‐derived NK cell starting material.
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