CTLL2 cells were collected, washed with fresh complete RPMI-1640 media and cultured with complete RPMI-1640 media free of IL-2 for 5 hours. After 5 hours, 1x105 CTLL2 cells were stained for 10 minutes with Zombie Aqua Fixable Viability stain (Biolegend, San Diego, CA). Cells were then stimulated for 15 minutes with AnnV-IL2 or IL-2 prepared in complete RPMI media using a 3-fold serial dilution. After 15 minutes, 1:1 volume of 8% PFA in PBS/3% MeOH was immediately added to each well and incubated at RT for 10 minutes. Cells were washed with MACS buffer (0.05% BSA in PBS) and resuspend with ice cold Perm III buffer (BD Biosciences, Franklin Lakes, NJ) and incubated overnight at -20°C. 5ul from each sample was collected for the isotype control. Cells were washed 2x with MACS buffer and stained with pSTAT5-Alexa647 for 30 minutes at 4°C. Cells were washed with MACS and samples acquired on a CytoFLEX S flow cytometer.
Zombie aqua fixable viability stain
Manufactured by BioLegend
Sourced in United States
The Zombie Aqua Fixable Viability stain is a fluorescent dye that can be used to assess cell viability. It is designed to detect dead or dying cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Fcr blocking antibody, by BioLegend (2 mentions)
Perm wash solution, by BioLegend (2 mentions)
Facsaria 2, by BD (2 mentions)
Perm buffer 3, by BD (1 mentions)
Facsymphony a1 cell analyzer, by BD (1 mentions)
Accutase, by Thermo Fisher Scientific (1 mentions)
Facsdiva software, by BD (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by BioLegend (1 mentions)
Facsaria 3, by BD (1 mentions)
Rneasy lysis buffer, by Qiagen (1 mentions)
Low protein binding microcentrifuge tubes, by Eppendorf (1 mentions)
7 protocols using zombie aqua fixable viability stain
1
Phospho-STAT5 Signaling Assay in CTLL2 Cells
2
Multiparametric flow cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dissociated cells were seeded on round-bottom 96-well plates and washed with 200 μl of ThermoFisher’s PBS buffer 1x (428 x g, 4 °C for 5 min). Twenty μl of Biolegend Zombie Aqua fixable viability stain (1:500) was added and incubated for 20 min in the dark at 4 °C. Excess viability stain was washed away with 200 μl of PBS/BSA/EDTA buffer (428 x g, 4 °C for 5 min) and the pellet resuspended and incubated for 10 min at 4 °C in 4 μl of Biolegend’s FcR blocking antibody (1:10). Cells were then incubated for 30 min at 4 °C with surface staining antibody master mix. After washing free unbound antibody with 200 μl of PBS (428 x g, 4 °C for 5 min), cells were fixed with 100 μl of Biolegend’s fixation buffer 1x for 20 min at 4 °C in the dark. Following fixation, cells were permeabilized and washed with 100 μl of Biolegend perm-wash solution 1 x (428 x g, 4 °C for 5 min) and the pellet resuspended and incubated for 30 min at 4 °C in intracellular staining antibody master mix (antibody dilution 1/100). Stained cells were placed in 200 μl of PBS/BSA/EDTA 1x and visualized on a BD Biosciences FACS Aria II using FACS Diva software. Analysis was performed using BD Biosciences FlowJo software. Antibody panels are described in supplemental material (Supplementary Table 2 ).
3
HBEC3KT Cell Viability Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HBEC3KT cells were removed from cell culture dishes using Accutase (ThermoFisher Scientific) and resuspended in 100μl 1×PBS. For viability, cells were incubated at room temperature for 10 min with 0.2 μl Zombie Aqua Fixable Viability stain (BioLegend) in 100 μl PBS. Control PE-mIgG2a, κ isotype (BioLegend, 400213), or PE-IL-28RA antibody (BioLegend, 337804) was added and incubated on ice for an additional 20 min. After incubation, the cells were washed with 1× PBS + 2% FBS and data were collected on a BD FACSymphony A1 Cell Analyzer (BD Biosciences) and analysis was performed using FlowJo software (FlowJo).
4
Endocytic Inhibition Modulates Monocyte Responses
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Freshly isolated PBMCs were left untreated, mock-treated with DMSO or treated with endocytic inhibitors in the absence of serum as above, then stimulated with 20 µg/mL pre-opsonized PGN in the presence of brefeldin A (3 µg/mL) for 6 h at 37 °C in a humidified atmosphere containing 5% CO2. After stimulation, cells were transferred to ice, stained for surface markers and viability (Zombie Aqua fixable viability stain, BioLegend, San Diego, CA, USA), fixed with 4% paraformaldehyde and stained for intracellular inducible antigens after saponin permeabilization. Dead cells were excluded based on viability stain positivity and monocytes were identified based on size, scatter and CD14 expression. Monocyte procoagulant responses were defined by tissue factor (TF/CD142) induction, while proinflammatory responses were defined by TNF. Data were collected on a LSRII cytometer using BD FACSDiva software (Version 9.0).
5
MRGX2 Expression Analysis by FACS
6
Isolation and Analysis of HSPC Subpopulations
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HSPC subpopulations were isolated using FACS as previously described.15 (link) For flow cytometric analysis of intracellular clusterin (CLU) and LGALS9 expression, the IntraPrep Leukocytic Permeabilization Reagent Kit (Beckman Coulter) was used. Dead cells were excluded in the analysis using Zombie Aqua Fixable Viability Stain (BioLegend). A list of all antibodies is provided in supplemental Table 2.
For MS analysis, 25 000 HSC/MPPs and CMP/MEPs were sorted into protein low-binding micro-centrifuge tubes (Eppendorf) and prepared for MS analysis. Details on sample preparation are provided in the supplemental Material. For RNA sequencing (RNA-seq) analysis, up to 10 000 HSC/MPPs, CMP/MEPs, CMPs, GMPs, and MEPs were sorted into RNeasy lysis buffer (Qiagen) containing β-mercaptoethanol. For the probes labelled PV1/8/12, CON1, CON2, and CON3, samples from different individuals had to be pooled to guarantee adequate HSC/MPP numbers for downstream MS and RNA-seq measurements (supplemental Table 1).
For MS analysis, 25 000 HSC/MPPs and CMP/MEPs were sorted into protein low-binding micro-centrifuge tubes (Eppendorf) and prepared for MS analysis. Details on sample preparation are provided in the supplemental Material. For RNA sequencing (RNA-seq) analysis, up to 10 000 HSC/MPPs, CMP/MEPs, CMPs, GMPs, and MEPs were sorted into RNeasy lysis buffer (Qiagen) containing β-mercaptoethanol. For the probes labelled PV1/8/12, CON1, CON2, and CON3, samples from different individuals had to be pooled to guarantee adequate HSC/MPP numbers for downstream MS and RNA-seq measurements (supplemental Table 1).
7
Dengue Virus Envelope Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were transferred to V-bottom 96-well plates and washed with 200 μl of PBS buffer 1× (428 × g, 4°C for 5 min). 20 μl of Zombie Aqua fixable viability stain (Biolegend, 1 : 500) was added and incubated for 20 min in the dark at 4°C. Excess viability stain was washed away with 200 μl of PBS/BSA/EDTA buffer (428 × g, 4°C for 5 min) and the cell pellet resuspended and incubated for 10 min at 4°C in 4 μl of FcR blocking antibody (Biolegend) (1/10). Cells were then incubated for 30 min at 4°C with surface staining antibody master mix (Supplementary Table 1 ). After washing free unbound antibody with 200 μl of PBS (428 × g, 4°C for 5 min), cells were fixed with 100 μl fixation buffer (Biolegend) 1× for 20 min at 4°C in the dark. Following fixation, cells were permeabilized and washed with 100 μl of perm-wash solution 1× (Biolegend, 428 × g, 4°C for 5 min) and the cell pellet resuspended and incubated for 30 min at 4°C with anti-DENV envelop protein antibody (clone 4G2 ATCC HB-112, labeled with Alexa Fluor 488) (Molecular probes; Thermo Fisher) (antibody dilution 1/100). Stained cells were resuspended in 200 μl of PBS/BSA/EDTA 1× and visualized on a BD Biosciences FACS Aria II using FACS Diva software. Analysis was performed using BD Biosciences FlowJo x10.0.7r2 software. Gating strategy is described in Supplementary Fig. 2 .
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!