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6 protocols using live dead fixable far red

1

CD8+ T-cell Mediated Cytotoxicity Assay

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A CD8+ T-cell line reactive to the HLA-A2-restricted peptide Influenza virus matrix protein MP58-66 (32% peptide-specific by tetramer staining) was thawed and rested for 3 h before use. Meanwhile, islet clusters were dissociated with TrypLE, stained with 1 μM CFSE or CellTrace Violet (CTV) and incubated for 2 h with 0.1 μM Influenza MP58-66 or peptide diluent, respectively. After washing, CFSE- and CTV-labelled SC-islets were mixed in equal numbers and cultured for 6 h in triplicate at 1 × 105 cells/each per well in 96-well flat-bottom plates, alone or with increasing numbers of T cells (0.2 × 105, 1 × 105, 5 × 105, and 10 × 105), corresponding to effector-to-target (E:T) ratios of 1:5, 1:1, 5:1, and 10:1, respectively. After washing, cells were stained with Live/Dead Fixable Far Red (Thermo Fisher), antibodies against HLA-A, B, C (RRID: AB_2566151) and PD-L1 (RRID: AB_940368) and acquired on a BD LSRFortessa flow cytometer. SC-islets were analysed by FlowJo after gating on Live/Dead¯ events and separation of CFSE+ (peptide-pulsed) and CTV+ (unpulsed) populations. Percent peptide-specific lysis at different E:T ratios is expressed as the ratio of live CFSE+/CTV+ cells normalized to the same ratio in wells containing SC-islet targets alone.
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2

Neutrophil Infection by Metacyclic Promastigotes

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Bone marrow derived neutrophils were isolated from C57/B6 mice using EasySep Mouse Neutrophil Enrichment Kit (STEMCELL, 19762) following manufacturer protocol. Metacyclic promastigotes were labelled with Carboxyfluorescein succinimidyl ester (CFSE) by incubating for 15 minutes at room temperature followed by wash. Isolated neutrophils were infected with CFSE stained metacyclic promastigotes at multiplicity of infection (MOI) of 3 metacyclic promastigotes per neutrophil. The infection was performed in the presence of tHDL and bHDL for four hours. After 4 hours, infected neutrophils were stained with Live-dead Fixable far red (ThermoFisher, L10120) and Ly6-G PE (BD Biosciences, 551461), fixed with PFA and assayed by flowcytometry.
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3

Multiparametric Flow Cytometry Analysis

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Human PBMC were stained in PBS with LIVE/DEAD Fixable Far Red (Thermofisher Scientific) for 15 min at room temperature. Human Fc‐block (BD Biosciences) was then added at 500 ng/test for a further 15 min at room temperature. Cells were washed once and stained in PBS + 2% FBS for 30 min at room temperature with anti‐surface marker mAb as listed in Supplementary table 1 and human MR1‐5‐OP‐RU or MR1‐Ac‐6‐FP BV421 tetramer (streptavidin‐BV421 from Biolegend). Cells were then washed twice before avidin and biotin blocking (Dako). Cells were then stained for a further 30 min at room temperature with human CD1d‐PBS44 BV605 tetramer (streptavidin‐BV605 from BD Horizon). Cells were washed twice and subsequently either subjected to intracellular transcription factor staining (see below) or fixed with 2% paraformaldehyde for 10 min at room temperature. Cells were analyzed immediately by flow cytometry using an LSRFortessa (BD Biosciences).
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4

Surface and Intracellular Cytokine Staining

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Cells were surface stained as described above with LIVE/DEAD Fixable Far Red (Thermofisher Scientific) and anti‐surface mAb above prior to fixation and permeabilization using 2% paraformaldehyde and 0.3% saponin (BD Biosciences). In brief, cells were fixed using 2% paraformaldehyde for 20 min at room temperature, washed once and then stained in 0.3% saponin overnight at 4 degrees with anti‐cytokine mAb as listed in Supplementary table 1. Cells were then washed twice with PBS and resuspended in PBS + 2% FBS, prior to flow cytometric analysis using an LSRFortessa (BD Biosciences).
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5

Characterizing cervical tissue-resident memory T cells

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Cervical tissue from healthy donors was obtained and digested, and the resulting cell suspension was stained for viability with Aqua vital dye (Invitrogen) and the following antibodies: CD69-FiTC (FN50 dilution 1/20, cat. no. 557049), CD45-PE (HI30, dilution 1/100, cat. no. 560975), CD3-PerCP (SK7, dilution 1/10, cat. no. 345766), CD19-V500 (HIB19, dilution 1/50, cat. no. 561121), PD1-BV421 (EH12.1, dilution 1/20, cat. no. 562516) (all from BD Biosciences), CD4-APC (OKT4 BioLegend, dilution 1/80, cat. no. 300514). AquaCD19CD45+ CD3+ CD4+ T cells were acquired in a BD FACSAriaTM II Cell Sorter and separated according to their CD69 expression into CD69+ (TRM) and CD69 (non-TRM). Sorted cells were then infected with 3655 TCID50 of the viral stock HIV-1BaL for 4 h at 37 °C or with medium in control conditions, washed with PBS and cultured in a 96-wells plate. After three days, cells were stained for viability with Live/Dead Fixable Far Red (Invitrogen), CD69-FiTC (FN50 dilution 1/20, cat. no. 557049) and HLA-DR-PerCP-Cy5.5 (G46-6, dilution 1/100, cat. no. 560652), before staining for p24 as described above. Fixed cells were finally acquired in a BD FACS Calibur flow cytometer and analyzed with FlowJo vX.0.7 software (TreeStar).
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6

PBMC Immunophenotyping Workflow

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PBMC were stained for extracellular markers (clones in parenthesis) with anti-CD2 (RPA-2.10, BioLegend, San Diego, CA, USA), anti-CD3 (BW264/56), anti-CD16 (3G8), anti-CD56 (REA196), anti-CD57 (TB03), anti-NKG2A (REA110), all from Miltenyi Biotec, Auburn, CA, USA and anti-NKG2C (134591, R&D Systems, Minneapolis, MN, USA). LIVE/DEAD Fixable Far Red (Invitrogen) was used to exclude dead cells. Fixation and permeabilization were performed using MACS Inside Stain Kit (Miltenyi Biotec) with polyclonal goat anti-human FcεRIγ (EMD Millipore, Etobicoke, ON, Canada), anti-IFN-γ (4S.B3), and anti-TNF-α (MAb11) from eBioscience, San Diego, CA, USA added for intracellular staining. The fluorescence minus one strategy was used to adjust multicolor compensation. Phenotypic analysis was performed using Kaluza Flow Cytometry Software 1.2 after data acquisition on a MoFlo Astrios EQ flow cytometer (both Beckman Coulter, Brea, CA, USA).
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