Following single-cell suspension preparation, cells were pelleted and subsequently stained with fluorophore-conjugated antibodies against CD11b (clone M1/70), CD45 (clone 30-F11), CD45.1 (clone A20), CD45.2 (clone 104), CX3CR1 (clone SA011F11; BioLegend), F4/80 (clone BM8; BioLegend), Ly6C (clone HK1.4), Ly6G (clone 1A8; BioLegend), Gr-1 (clone RB6-8C5), CD115 (clone AFS98), MHC II (clone M5/114.15.2), CD11c (clone N418), CD64 (clone X54-5/7.1; BioLegend), CD86 (clone GL1; BioLegend), Tmem119 (clone 106–6; Abcam), and either DAPI or propium iodide viability dyes (all from eBioscience if not indicated otherwise). Flow cytometry was performed using a Fortessa analyzer (BD Biosciences), and FACS was performed using a FACS Aria II (BD Biosciences) or LSRII (BD Biosciences). The gating strategies used for flow cytometry analysis of embryonic tissues are adapted from Hoeffel et al. (2015) (link) and shown in Fig. S5. Resident microglia were sorted as doublet−DAPI−CD11b+CD45int (Fig. S2 B). Flow cytometry data analysis was performed using FlowJo (TreeStar) software. For ULI RNA-seq, microglia were double-sorted to reach a purity of >98%, and 1,000 cells were sequenced.
Tmem119 clone 106 6
Manufactured by Abcam
Tmem119 (clone 106-6) is a protein marker that is specifically expressed in microglia, the resident immune cells of the central nervous system. It serves as a reliable identifier of microglial cells.
Automatically generated - may contain errors
Lab products found in correlation
Fortessa analyzer, by BD (1 mentions)
Gr 1 clone rb6 8c5, by BioLegend (1 mentions)
Cd64 clone x54 5 7, by BioLegend (1 mentions)
Cd86 clone gl 1, by BioLegend (1 mentions)
Cd11b clone m1 70, by BioLegend (1 mentions)
F4 80 clone bm8, by BioLegend (1 mentions)
Cd45 clone 30 f11, by BioLegend (1 mentions)
Facsaria 2, by BD (1 mentions)
Cell strainer cap, by STEMCELL (1 mentions)
Facs staining buffer, by BD (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
2 protocols using tmem119 clone 106 6
1
Sorting and Sequencing Microglia from Embryonic Tissues
2
Multicolor flow cytometry of microglia
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All cells were stained with the following antibodies: FITC-conjugated CX3CR1 (clone SA011F11, BioLegend), Tmem119 (clone 106–6, Abcam), APC-Cy™7-conjugated CD11b (clone M1/70, BD Biosciences), and PE mouse anti-rabbit IgG (BD Biosciences). The incubation of all antibodies was performed for 15 min at 4 °C in the dark, and then centrifugation was carried out at 1500 rpm and 4 °C using a bench-top centrifuge. FACS staining buffer was used in the washing procedures of this experiment (BD Pharmigen™, New Jersey, USA). Afterwards, the cells were then filtered using a round-bottom tube with a cell strainer cap (STEMCELL Technologies). Cell data were acquired on an LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software (FlowJo LLC).
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