Microglia cells were isolated via magnetic-activated cell sorting (MACS) using mouse anti-CD11b magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer’s instructions, with some modifications. The MACS buffer used consisted of 2% bovine serum albumin (BSA) diluted in DPBS from a 7.5% cell culture-grade BSA stock (Thermo Fisher Scientific Inc., Waltham, MA, USA). Total hippocampal/frontal cortex cell pellets after percoll separation (see above) were re-suspended in 90 μL MACS buffer and 10 μL anti-mouse-CD11b magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). The cells were then incubated for 15 min at 4 °C. Cells were washed with 1 mL MACS buffer and pelleted at 300 rcf for 5 min at 4 °C. The cells were then passed through an MS MACS column (Miltenyi Biotec, Bergisch Gladbach, Germany) attached to a magnet. After washing the columns three times with MACS buffer, microglia were flushed from the column with 1 mL of MACS buffer and pelleted at 300 rcf for 5 min at 4 °C. Cell pellets were then snap-frozen in liquid nitrogen and stored at −80 °C.
Anti mouse cd11b magnetic beads
Manufactured by Miltenyi Biotec
Sourced in Germany
Anti-mouse CD11b magnetic beads are a laboratory product used for the isolation and enrichment of CD11b-positive cells from mouse samples. The beads are coated with antibodies specific to the CD11b surface marker, allowing for the magnetic separation of CD11b-expressing cells from a heterogeneous cell population.
Automatically generated - may contain errors
Lab products found in correlation
Macs buffer, by BD (3 mentions)
Cell strainer, by BD (2 mentions)
Papain solution, by Worthington (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Macs ms column, by Miltenyi Biotec (1 mentions)
Adult brain dissociation kit for mouse and rat, by Miltenyi Biotec (1 mentions)
Fitc conjugated anti mouse f4 80, by Thermo Fisher Scientific (1 mentions)
Pe conjugated anti mouse cd11c, by Thermo Fisher Scientific (1 mentions)
Flow cytometry, by BD (1 mentions)
6 protocols using anti mouse cd11b magnetic beads
1
Microglia Isolation via MACS
2
Isolation and Enrichment of Hippocampal Microglia
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Hippocampi from 3 animals were pooled and digested into a single cell suspension by using an Adult Brain Dissociation Kit for mouse and rat (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) according to the manufacturer’s manual. After Percoll separation, the cells were washed in ice-cold MACS-buffer (PBS, 0.5% bovine serum albumin, 2 mM EDTA) and stained with anti-mouse CD11b magnetic beads (Miltenyi Biotech) at 4 °C for 20 min. Due to limitation of cell number, six hippocampi from 3 animals were pooled and total cells were passed through large-sized MACS columns (Miltenyi Biotech). The flow through was discarded and the cells were flushed out of the column in MACS buffer, pulled down by centrifugation and resuspended in RIPA buffer (Thermo Fisher Scientific) for Western blotting or 2M urea buffer for microglia specific proteomics, and stored at −80 °C.
3
Isolation and Characterization of M1/M2 Macrophages
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The retinas of OIR mice treated with PBS or PDTC were digested in preheated papain solution (Worthington Biochemical Corp., Lakewood, NJ, USA) for 30 minutes. The obtained cell digestion suspension was filtered, centrifuged, and suspended with MACS buffer (BD Biosciences, San Jose, CA, USA). After incubation with anti-mouse CD11b magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) at 4°C for 20 minutes, these cells were screened with pre-humidified mass spectrometry column (BD Biosciences). Collected CD11b+ cells were incubated with FITC-conjugated anti-mouse F4/80 (eBioscience), PE-conjugated anti-mouse CD11c (eBioscience), Alexa Fluor 647-conjugated CD206 (Bio-Rad AbD Serotec, Ltd., Oxford, UK), and the matching control isotype IgG (MCA421; Bio-Rad AbD Serotec) at 4°C for 30 minutes. After washing and resuspension, the cells were analyzed by flow cytometry (BD Biosciences). F4/80+/CD11c+/CD206– (F4/80+ and CD11c+) cells were identified as M1 polarized macrophages, and F4/80+/CD11c–/CD206+ (F4/80+ and CD206+) cells were identified as M2 polarized macrophages as previously described.6 (link),20 (link),21 (link) Data analysis was performed by FlowJo software (BD Life Sciences, Franklin Lakes, NJ, USA).
4
Isolation of Retinal Cells Post-Intravitreal Injection
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After intravitreal injection with 1 μg/μl AC220 (or control vehicle), mouse retinas at P15, P18, and P21 (6–10 retinas per group) were obtained for the isolation of retinal cells as described previously, using antimouse CD11b magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) and a premoistened MS column (BD Biosciences) [16 (link)].
5
Isolating CD11b+ Cells from Mouse Retinas
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The retinas of normal mice and those with OIR were carefully dissected out and digested in pre-warmed 16.5 U/ml papain solution (Worthington, Freehold, NJ, USA) for 30 min with gentle pipetting, and the cell digestion suspension was then transferred and passed through cell strainers (BD Falcon, Franklin Lakes, NJ, USA) to obtain single cell suspension. The cells were spinned down at 900 rpm. After gently removing the supernatant, the cell pellet was suspended with 90 µl MACS buffer (BD Biosciences, San Jose, CA, USA), mixed well with 10 µl anti-mouse CD11b magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany), incubated at 4°C for 20 min, washed once and resuspended in 500 µl MACS buffer and loaded on a pre-moistured MS column (BD Biosciences, San Jose, CA, USA) to sort for CD11b+ cells according to the manufacturer's instructions. The selected cells were collected and proceeded to flow cytometric analysis.
6
Isolation of Microglia from Mouse Retinas
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Mouse retinas of P15, P17 and P21 from wild-type (WT) and OIR mice were carefully dissected out and digested in pre-warmed 16.5-U/ml papain solution (Worthington Biochemical) for 30 min with gentle pipetting. Trypsin inhibitor was added to stop digestion. Then the cell digestion suspensions were transferred and passed through cell strainers (BD Falcon) to ensure single-cell suspension, and cells were spun down at 900 rpm (89 g). After gently removing the supernatant, the cell pellet was resuspended with 90 μl MACS buffer (BD Biosciences) and mixed well with 10 μl anti-mouse CD11b magnetic beads (Miltenyi Biotec), incubated at 4 °C for 20 min, washed once, and resuspended in 500 μl MACS buffer. The cell pellet was then loaded on a premoisturized MS column (BD Biosciences) and washed twice. Then the columns were taken off the magnetite and residue cells were flushed out of the column, according to the manufacturer’s protocol. The selected cells were collected and analyzed by flow cytometry. All retina cell samples from different mice were treated as mentioned separately.
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