Single-cell suspensions were stained with fluorescently conjugated antibodies in a 1:100 dilution unless otherwise noted. For live/dead discrimination, cells were stained with either LIVE/DEAD Blue Reactive Dye (Invitrogen) (20 minutes, on ice, before primary) or Alexa Fluor NHS Ester (ThermoFisher) (with primary stain), and 4μg/mL anti-CD16/32 (2.4G2; Bioxcell) to block Fc receptors. The following anti-mouse antibodies were used, with clone and source company listed: CD45 PerCP-Cyanine5.5 (30-F11, BioLegend), CD45 BV510 (30-F11, BioLegend), CX3CR1 BV605 (SA011F11, BioLegend), F4/80 BV711 (BM8, BioLegend), Ly6G PerCP-Cyanine5.5 (1A8, BioLegend), Ly6C PE/Cyanine7 (HK1.4, BioLegend), CD11b BUV395 (M1/70, BD Horizon), CD11c BV650 (N418, BioLegend), Ki67 PE/Dazzle (16A8, BioLegend). Intracellular Ki67 was stained using the eBioscience Transcription Factor Staining Kit, following the included protocol. The BD FACS ARIA II was used for cell sorting, and the Bio-Rad ZE5 Yeti was used for flow cytometry analysis. Data analysis was performed using FlowJo v10 (FlowJo LLC).
Ze5 yeti
Manufactured by Bio-Rad
The ZE5 Yeti is a compact, benchtop flow cytometer designed for multi-parameter analysis of cells and particles. It features a simple user interface, automated setup, and reliable performance for a variety of applications.
Automatically generated - may contain errors
Lab products found in correlation
Facsaria 2, by BD (1 mentions)
Alexa fluor nhs ester, by Thermo Fisher Scientific (1 mentions)
Cd45 bv510 30 f11, by BioLegend (1 mentions)
Transcription factor staining kit, by Thermo Fisher Scientific (1 mentions)
Cd45 percp cyanine 5, by BioLegend (1 mentions)
Anti cd16 32 2.4g2, by BioXCell (1 mentions)
Ki 67 pedazzle, by BioLegend (1 mentions)
F4 80 bv711, by BioLegend (1 mentions)
Cd11c bv650, by BioLegend (1 mentions)
Alexa fluor 405 nhs ester, by Thermo Fisher Scientific (1 mentions)
2 protocols using ze5 yeti
1
Multiparameter Flow Cytometry Protocol
2
Isolation and Labeling of Photoreceptor Outer Segments
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Photoreceptor outer segments (POSs) were isolated from fresh porcine eyes (Sierra for Medical Science, http://www.sierra-medical.com ) as previously described (60 (link)) and conjugated with Alexa Fluor 405 NHS Ester (Invitrogen, Thermo Fisher Scientific, A30100) as described in the product manual. fPOS preparations were quantified using Bradford assays. fPOS aliquots were stored at –80°C. Cells were treated with 10 μg/cm2 fPOS and incubated at 37°C for 5 hours. All media were removed, and cells were rinsed with Dulbecco’s PBS (–/–) and then treated with trypsin for 5 minutes at 37°C. Cells were dissociated in trypsin and then diluted 1:1 in FACS buffer (1× PBS [–/–], 2.5 mM EDTA, 25 mM HEPES, pH 7.0, 1% heat-inactivated FBS, 1:2,000 DRAQ5). Samples were immediately analyzed on a ZE5 YETI (Bio-Rad). Cells were gated according to DRAQ5 labeling, and 40,000 events were collected per sample. Data were processed using FlowJo software (BD Biosciences).
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