2.5 × 104-irradiated T2 cells loaded with 10 μM peptide and 5 × 104 bulk-transduced T cells labeled with Cell Trace Violet (Invitrogen) were co-cultured for 5 days in round-bottom 96-well plates containing 250 μl of culture medium per well. Cells from duplicate wells were pooled, stained for CD8 and CD19, and analyzed by flow cytometry. Data were acquired using an LSRFortessa and analyzed with FlowJo software.
Lsrfortessa
Manufactured by FlowJo
Sourced in United States
The LSRFortessa is a flow cytometry instrument designed for advanced cell analysis. It features multiple laser configurations and a range of fluorescence detection channels to enable comprehensive multiparametric analysis of diverse cell samples. The LSRFortessa provides researchers with a powerful tool for high-performance flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsr 2, by BD (5 mentions)
Countess, by Thermo Fisher Scientific (3 mentions)
Fixation permeabilization kit, by Thermo Fisher Scientific (3 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (3 mentions)
70 μm cell strainer, by Thermo Fisher Scientific (3 mentions)
Dnase 1, by Roche (3 mentions)
Collagenase d, by Roche (3 mentions)
Ti2 inverted microscope, by Nikon (2 mentions)
Cytof software v7, by Standard BioTools (2 mentions)
Hyperion imaging system, by Standard BioTools (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Celltrace violet, by Thermo Fisher Scientific (2 mentions)
Rbc lysis buffer, by BioLegend (2 mentions)
Annexin 5 binding buffer, by BD (1 mentions)
Annexin 5 fitc, by BD (1 mentions)
Foxp3 efluor450, by Thermo Fisher Scientific (1 mentions)
Aqua viability dye, by Thermo Fisher Scientific (1 mentions)
Cd8 qdot 605, by Thermo Fisher Scientific (1 mentions)
Cd4 qdot655, by Thermo Fisher Scientific (1 mentions)
Pd 1 pe, by BD (1 mentions)
48 protocols using lsrfortessa
1
T cell Co-culture Assay
2
CD36 Expression Analysis by Flow
3
Apoptosis Assay for LN229 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LN229 cells were seeded 1 × 106 cells in a 10 cm dish. After 56 h of drug treatment, media were removed and placed in a labeled 15 mL falcon tube. Cells were collected through trypsin dissociation, added to the 15 mL tube, and washed twice with PBS without calcium or magnesium. Cell numbers were counted on Countess (Thermo Fisher). Cells were pelleted at 500× g for 10 min. The cell pellet was resuspended in Annexin V Binding Buffer (BD Pharmingen, 556454 Franklin USA), and 5 × 105 cells were transferred to a FACS tube containing 5 µL propidium iodide (PI) staining solution (Peprotech 60910-00) and 5 µL FITC-Annexin V (BD Pharmingen, 556419). Cells were incubated for 15 min in the dark. After incubation, 400 µL of Annexin V Binding Buffer was added and FACS was performed on a BD LSRII or LSR Fortessa and analyzed using FlowJo v10. Compensation controls used for this analysis included unstained cells and cells stained for each individual fluorophore (Cy5-PI and FITC-Annexin V). Gating was applied to eliminate cell debris and doublets through forward and side scatter plots.
4
Comprehensive Immune Profiling of Pembrolizumab Therapy
5
Comprehensive Flow Cytometry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At least 5000 events of the flow cytometry‐gated populations were collected. Flow cytometry data were acquired on BD LSRFortessa and analyzed using FlowJo V10 software. Statistical tests were performed using GraphPad Prism 8.0.2 and described in respective figure legends. P‐value < 0.05 was determined to be significant. Overall results were assessed based on reproducible statistical trends across at least three independent experiments.
6
EphA2 expression analysis by flow cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Surface expression of EphA2 was determined by flow cytometry analysis. A total of 1 × 106 WT HEK293T cells, EphA2- or EphA4 single-knockout, or double-knockout cells were harvested, washed with PBS containing 1% bovine serum albumin (BSA), and incubated with 5 μl of phycoerythrin (PE)-conjugated EphA2 antibody (BioLegend, SHM16) in 50 μl PBS containing 1% BSA for 30 min at 4°C. Cells were then washed and diluted in 300 μl PBS containing 1% BSA. Data were acquired using a BD LSRFortessa instrument, and the FlowJo software was used for analysis.
7
Isolation and Cloning of Single Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells in culture were collected and resuspended in PBS with 2% FBS. Cells were stained with Fc receptor blocking antibody (cat. #101319, BioLegend) for 5 minutes at room temperature, then the cells were stained for cell surface markers using fluorochrome-coupled antibodies (1:100 dilution; Supplementary Table S2) on ice for 30 minutes. Cells were washed twice using PBS with 2% FBS. Well stained cells were collected by BD Biosciences LSRFortessa and the data were analyzed with FlowJo software. All the samples were run in triplicates. For cell sorting, well stained cells were re-suspended in culture medium and collected by SY3200 cell sorter and the data were analyzed with FlowJo software. For generating single cell clone, cells in culture were collected and filtered through 40-μmol/L cell strainers (cat. #08–771–1, Thermo Fisher Scientific) to obtain single-cell suspensions. Suspended cells with or without surface marker staining depending on experiment design were sorted by SY3200 cell sorter to distribute single-cell clone to 96-well plate with 100 μL culture medium. Each single-cell clone was expanded for further experiments.
8
Mitochondrial ROS Measurement in LECs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Control and shunt LECs were incubated with MitoSOX Red added at a final concentration of 5 mmol/L. Stained LECs were analyzed on a BD LSRFortessa flow cytometer and using FlowJo V10.3.0 software.
9
Comprehensive Single-cell Immunophenotyping
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single-cell suspension prepared from tumor tissue were pre-incubated with anti-CD16/32 monoclonal antibody (FcR-blocking, BD Biosciences, San Jose, California, USA, clone 2.4G2) and then stained with antibodies against membrane markers for 45 min at 4℃. Dead cells were marked using Fixable Viability Stain 510 (BD Pharmingen, San Diego, California, USA, No. 564406). For intracellular staining, cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience, San Diego, California, USA, No. 00-5523-00) or the Intracellular Fixation & Permeabilization Buffer Set (eBioscience, No. 88-8824-00) following the manufacture’s instruction. Antibodies were added and incubated for 1 hour at 4℃. Intracellular cytokine staining was performed 4–6 hours after ex vivo stimulation with leucocyte activation cocktail in the presence of GolgiStop (BD Pharmingen, No. 550583) at 37℃. All the fluorescently labeled antibodies used for staining are listed in online supplemental table S1 . Data were collected with a BD LSRFortessa and analyzed using FlowJo (V.10.0). According to the isotype and fluorescence-minus-one, the gating strategies for flow cytometry were showed in online supplemental figure S4 .
10
Terminal Erythroid Differentiation Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Terminal erythroid differentiation was monitored using CD44, Ter119, and forward scatter as described29 (link) and analyzed using a LSRFortessa and FlowJo v9.9.4 software. Cell cycle and apoptosis were analyzed using BD Bioscience’s APC BrdU Flow Kit and Annexin V56 (link), respectively, per manufacturer’s instructions.
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!