Male and female Mobp-TDP43 and Mog-TDP43 mice aged at P30 were cardiac perfused with ice-cold 1× Hank's Balanced Salt Solution (HBSS) w/o Ca2+ and Mg2+. Only the cortex was dissected out for downstream processing and mechanically dissociated with a razor blade on ice. The Miltenyi Neural Tissue Dissociation kit was used to perform enzymatic dissociation of the tissue into a single-cell suspension, and Myelin Debris Removal solution (Miltenyi) to remove myelin debris. The cells were resuspended in FACS sorting buffer (5 ml of 1 M HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) + 2 ml of Fetal Bovine Serum (FBS) + 800 μl of 0.5 M EDTA (Ethylenediaminetetraacetic acid) in 200 ml HBSS w/o Ca2+, Mg2+) and filtered through 35 μm strainer. The cells were sorted for endogenous GFP fluorescence on BD FACS Aria IIu Cell Sorter using a 100 μm nozzle at the Ross Flow Cytometry Core Facility at Johns Hopkins University School of Medicine.
Myelin debris removal solution
Manufactured by Miltenyi Biotec
Sourced in Germany
The Myelin Debris Removal solution is a laboratory product designed to facilitate the removal of myelin debris from cell suspensions. It is a specialized tool used in various research applications involving the study of neural or glial cells.
Automatically generated - may contain errors
Lab products found in correlation
Neural tissue dissociation kit, by Miltenyi Biotec (2 mentions)
Facsaria iiu cell sorter, by BD (2 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Macs neural tissue dissociation kit with papain, by Miltenyi Biotec (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Single cell rnaseq kit, by Tecan (1 mentions)
3 protocols using myelin debris removal solution
1
Isolation of GFP-positive Cells from Mobp-TDP43 and Mog-TDP43 Mice
2
Brain Immune Cell Isolation Protocol
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Mice were anesthetized with isoflurane and pentobarbital before being perfused with ice-cold phosphate-buffered saline (PBS) containing heparin. Brains were removed and the hemispheres were separated before being placed into ice-cold Hanks’ balanced salt solution without Ca2+ and Mg2+ (HBSS, Life Technologies, Grand Island, NY). Tissue was enzymatically and mechanically dissociated using a MACS Neural Tissue Dissociation Kit with Papain (Miltenyi Biotec, Bergisch Gladbach, Germany) and then treated with a myelin debris removal solution (Miltenyi Biotec). Isolated brain immune cells were used for either flow cytometer analyses or cultured for ex vivo phagocytic activity assays.
3
Isolation and Sequencing of Oligodendrocytes
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Male and female Mobp-TDP43 and Mog-TDP43 mice aged at P30 were cardiac perfused with ice-cold 1x HBSS w/o Ca 2+ and Mg 2+ . Only the cortex was dissected out for downstream processing and mechanically dissociated with a razor blade on ice. The Miltenyi Neural Tissue Dissociation kit was used to perform enzymatic dissociation of the tissue into a single cell suspension, and Myelin Debris Removal solution (Miltenyi) to remove myelin debris. The cells were resuspended in FACS sorting buffer (5 mL of 1 M HEPES + 2 mL of FBS + 800 μl of 0.5 M EDTA in 200 mL HBSS w/o Ca 2+ , Mg 2+ ) and filtered through 35 μm strainer. The cells were sorted for endogenous GFP fluorescence on BD FACS Aria IIu Cell Sorter using a 100 μm nozzle at the Ross Flow Cytometry Core Facility at Johns Hopkins University School of Medicine.
RNA isolation, cDNA library preparation, and sequencing RNA was isolated from FACS purified oligodendrocytes using QIAGEN RNeasy Micro Kit following the manufacturer's instructions. cDNA was synthesized by using QIAGEN QuantiTect Reverse Transcription Kit.
Due to low amount of cDNA, Nugen Single-cell RNAseq kit was used to generate cDNA libraries for 100 bp paired-end sequencing on Illumina HiSeq (70-100 million paired reads/sample).
RNA isolation, cDNA library preparation, and sequencing RNA was isolated from FACS purified oligodendrocytes using QIAGEN RNeasy Micro Kit following the manufacturer's instructions. cDNA was synthesized by using QIAGEN QuantiTect Reverse Transcription Kit.
Due to low amount of cDNA, Nugen Single-cell RNAseq kit was used to generate cDNA libraries for 100 bp paired-end sequencing on Illumina HiSeq (70-100 million paired reads/sample).
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