The acceptance criteria for F-TIL included a viability assay, trypan blue exclusion, measured via TC20 Automated Cell Counter (BioRad). The acceptance is set at not less than (NLT) 70% viable. Also, cell density and cell count were determined from this measurement with an accepted cell count of NLT 1×109 cells. The F-TIL identity and potency were established from an interferon gamma (IFNɣ) release assay. IFNɣ+ T cells were assayed by intracellular staining with and without soluble CD3 (OKT3, Miltenyi Biotec) stimulation. Briefly, after control or OKT3 stimulation with Golgi Plug and Golgi Stop (Miltenyi Biotec), cells were pelleted and stained with Viobility 405/520 (Miltenyi Biotec), followed by CD3-VioBlue, CD4-FITC, and CD8-PE (all from Miltenyi Biotec). Lastly, cells were treated with Inside Stain (Miltenyi Biotec) and then stained with anti-IFNγ-APC (Miltenyi Biotec). Cellular preparations were assayed using a Miltenyi MACSQuant 10 analyzer.
Viobility 405 520
Manufactured by Miltenyi Biotec
The Viobility 405/520 is a multicolor flow cytometry reagent that enables the simultaneous detection of two cellular parameters. It is designed to provide bright and stable fluorescence signals for cell analysis applications.
Automatically generated - may contain errors
Lab products found in correlation
Tc20 automated cell counter, by Bio-Rad (1 mentions)
Macsquant10 analyzer, by Miltenyi Biotec (1 mentions)
Cd8 pe, by Miltenyi Biotec (1 mentions)
Inside stain, by Miltenyi Biotec (1 mentions)
Anti ifnγ apc, by Miltenyi Biotec (1 mentions)
Cd3 vioblue, by Miltenyi Biotec (1 mentions)
Cd4 fitc, by Miltenyi Biotec (1 mentions)
Lsr fortessa cytometer, by BD (1 mentions)
Anti cd3 bv510, by BD (1 mentions)
Cd56 bv510, by BioLegend (1 mentions)
Hla dr vioblue, by Miltenyi Biotec (1 mentions)
Anti cd14 bv650, by BioLegend (1 mentions)
Cd19 viogreen, by Miltenyi Biotec (1 mentions)
Cd16 pe, by Beckman Coulter (1 mentions)
2 protocols using viobility 405 520
1
F-TIL Viability, Identity, and Potency Assays
2
Phenotypic analysis of dendritic cells
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Frozen PBMC were thawed in RPMI-50% FCS, counted in Trypan blue, and 1 × 106 cells were stained with a viability dye (Viobility 405/520, Fixable Dye, Miltenyi-Biotech), Lineage antibodies (anti-CD3-BV510 (BD Biosciences), CD19-VioGreen (Miltenyi-Biotech) and CD56-BV510 (BioLegend)), and anti-CD14-BV650 (Biolegend), HLA-DR VioBlue (Miltenyi-Biotech), CD141-BV785 (BioLegend), CD16-PE (Beckman-Coulter), CD1c-BV605 (BD Biosciences), FcERI-PerCP-Vio700 (Miltenyi-Biotech), CD123-APC-Vio770 (Miltenyi-Biotech), CD11c-PE-Vio615 (Miltenyi-Biotech) antibodies (all antibodies are described in Additional file 1 : Table S3). Samples were processed on the BD LSRFortessa cytometer (BD Biosciences). pDC and DC cells were identified as described by Mair et al. [49 (link)]. Data were analyzed using FlowJoTM version v10·8 software (BD Life Sciences).
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