LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit, for UV excitation

Protocol tips

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	Add reconstituted fluorescent reactive dye and incubate at room temperature or on ice for 30 minutes

Protocol tips
Add reconstituted fluorescent reactive dye and incubate at room temperature or on ice for 30 minutes

Publication protocol

CD11b+-sorted cells were collected from the astrocyte feeder cells using MACs (Miltenyi). DAPI was used to determine LIVE/DEAD cells. Next, the cells were washed and stained with primary antibodies such as Ms anti-human anti-CD11b (1:200) (abcam, Cat # ab91145), and HLA-DR (1:200) (abcam, Cat # [CR3/43] ab7856), rabbit pAB anti-CD45 (1:200) (abcam, ab10558), mouse mAB anti-CD86 (1:200) (abcam, Cat # [BU63] ab23556), rabbit anti-human Iba1 (1:100) (Wako, Cat # 019-19741), CD80 (1:200) (abcam), rabbit pAB CX3CR1 (1:200) (abcam, Cat # ab8021), goat pAb TREM-2 (1:200) (abcam, Cat # ab85851) antibodies, and their corresponding isotype controls for 1 hr at 4°C. After incubation, the cells were washed and stained with corresponding secondary antibodies either tagged with corresponding Alexa fluor 488 or Alexa fluor 546 for 45 min on ice, and subsequently the signals were analyzed using Moflo Astrios Cell Sorter (Beckman Coulter, Inc). For mouse iPS-MG, CD11b+-sorted cells were collected from the astrocyte feeder cells using MACs (Miltenyi). Subsequently, the cells were incubated with Fc receptor block consisting of Rat anti-mouse CD16/32 (1:100) (BD Pharmingen, Cat # 553142) and Mouse IgG (1:50) (Jackson Immunoresearch) for 15 min at 4°C. Staining was carried out for the dead cells using LIVE/DEAD® Fixable Blue Dead Cell Stain Kit for UV excitation (Molecular Probes) for 30 min at 4°C. Next, the cells were washed and stained with fluorochrome-conjugated Rat anti-mouse Ly6C-APC-Cy7 (1:400) (Biolegend, Cat # 128026, clone # HK1.4), Rat anti-mouse Ly6G-PE (1:200) (BD Pharmingen, Cat # 551461, clone # 1A8), Rat anti-mouse CD45-PerCP-Cy5.5 (1:50) (BD, Cat # 550994, clone # 30-F11 ), Rat anti-mouse CD39-PE-Cy7 (1:200) (eBioscience, Cat # 25-0391-80, clone # 24DMS1), Rat anti-mouse Thy1.2-Alexa fluor 700 (1:1200) (Biolegend, Cat # 105320, clone # 30-H12), Anti-mouse NK1.1-PE (1:200) mAb (eBioscience, Cat # 12-5941-83, clone # PK136), Rat anti-mouse B220-APC (1:1000) (BD Pharmingen, Cat # 553092, clone # RA3-6B2) antibodies, and their corresponding isotype controls for 1 hr at 4°C. All antibodies used are commercially validated. After incubation, the cells were washed twice and fixed with 1% paraformaldehyde. The signals were analyzed using a LSR II flow-cytometer (BD).

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