To assess the oxidative burst of neutrophils, we operated dihydrorhodamine123 (DHR123, Sigma Aldrich, Darmstadt, Germany) and phorbol 12-myristate 13-acetate (PMA) (Sigma Aldrich, Darmstadt, Germany). In all, 500 µL of peripheral blood in EDTA was RBC lysed, washed, and incubated with 5 pM of DHR for 5 min at 37 °C in a 5% CO2 atmosphere. Then, we added 50 nM of PMA and left the mixture for 15 min at 37 °C. Afterward, samples were incubated with antibodies against CD15 (APC, BD Biosciences, San Diego, CA, USA) and CD10 (APC-Cy7, ExBio) for 20 min at room temperature. Cells were washed and fixed in solution, and LSR Fortessa flow cytometry (BD Biosciences, San Diego, CA, USA) was performed. Approximately 500,000 events were obtained and analyzed with the FlowJo™ software (v.10.8.1).
Lsrfortessa flow cytometry
Manufactured by BD
Sourced in United States
The BD LSRFortessa is a flow cytometry instrument that enables the analysis of cells or particles in a fluid sample. It is designed to measure various characteristics of cells, including size, granularity, and the expression of specific markers on their surface or within the cells. The BD LSRFortessa is capable of detecting multiple fluorescent signals simultaneously, allowing for the simultaneous analysis of multiple parameters.
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Lab products found in correlation
Eflour450, by Thermo Fisher Scientific (2 mentions)
Annexin 5 fitc propidium iodide apoptosis detection kit, by Thermo Fisher Scientific (2 mentions)
Dhr123, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Cd41 clone mwreg30, by BD (1 mentions)
Ter119, by BD (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Annexin 5 fitc, by Thermo Fisher Scientific (1 mentions)
7 aminoactinomycin d, by BD (1 mentions)
Rabbit igg monoclonal isotype control, by Abcam (1 mentions)
Cd16 cd32 clone 2.4g2, by BD (1 mentions)
Facsaria 2 sorter, by BD (1 mentions)
Cd3e clone 145 2c11, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 anti rabbit igg, by Cell Signaling Technology (1 mentions)
Cycletest plus dna reagent kit, by BD (1 mentions)
Mouse th1 th2 th17 cytokine kit, by BD (1 mentions)
Pe cyanine7 anti mouse cd4, by BioLegend (1 mentions)
Live dead fixable dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Anti b220 percp cy5, by BD (1 mentions)
Pe anti mouse cd138 syndecan 1, by BioLegend (1 mentions)
30 protocols using lsrfortessa flow cytometry
1
Neutrophil Oxidative Burst Assessment
2
Multiparametric Flow Cytometry of Mouse Hematopoiesis
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Flow cytometry was performed using the LSRFortessa Flow Cytometry (BD Bioscience), and the data analysed using FlowJo v7.6.5 (TreeStar) Software. The mouse antibodies TER119 (BD Bioscience), CD71 (clone RI7217, Biolegend), CD41 (clone MWReg30, BD Bioscience) and CD42a (clone Xia.B4, Emfret Analytics) were used at the concentrations recommended by the manufacturers.
3
Flow Cytometry Analysis of Immune Cells
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All gating strategy information is depicted in the Supplementary Fig. 8 . Single cell suspensions from the indicated tissues were prepared as described above. Cells were resuspended in staining buffer (PBS, 1% FBS, 1 mM EDTA), followed by blocking of the Fc receptor and staining with the indicated antibodies for 30 min on ice in the dark. All antibodies used for flow cytometry (listed in Supplementary Table 2 ) were diluted as 1:200. For intracellular staining, cells were stained according to recommendations of the manufacturer of the eBioscience Foxp3 / Transcription Factor Staining Buffer Set (Cat No: 00-5523-00). Cells were washed and resuspended in staining buffer or PBS and either analyzed on LSRFortessa flow cytometry (BD Biosciences) or Canto flow cytometry (BD Bioscience). Data were analyzed with FlowJo V10.7.1(BD) software.
4
Dil-EMs Cytometry Evaluation
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MSCs and C28/I2 cells were treating with Dil-EMs for 2 h. Then the cell suspensions were collected and tested using a BD LSRFortessa Flow Cytometry. Flow data were then analyzed using FlowJo software.
5
Evaluation of Cellular Death in EPN and hAFSCs
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The cellular death was evaluated in EPN and hAFSCs cells incubated with DMEM-LG (negative control), Amblyomin-X [10 µM or 20 µM], Cisplatin [20 µM] for 24 or 48 h. In addition, as positive control, the cells were incubated with H2O2 [30%] for 30 minutes. Amounts of 105 EPNs cells or hAFSCs were twice washed with cold PBS. To labeling procedure, the cells were resuspended in 100 µL of Binding Buffer 1× (Molecular Probes, Invitrogen, USA), and were added FITC Annexin V (Molecular Probes, Invitrogen, USA) and 7-Amino-Actinomycin D (AAD – BD Biosciences), 5 µL each. The samples were incubated during 15 minutes in the dark at room temperature. Posteriorly, the volumes were adjusted to 500 µL using Binding Buffer 1×. Data acquisition was performed within one hour of labeling in LSRFortessa flow cytometry (BD Biosciences, San Jose, CA, USA) and analyzed by the software FlowJo.
6
In Vivo Cytotoxicity Assay
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A single-cell suspension of splenocytes from naïve syngeneic mice was diluted to 1.5 × 108/ml in RPMI1640 containing 10% FBS and 2% penicillin and streptomycin pulsed at 37°C with or without 5 μg/ml peptides as described previously [19 (link)]. After 4 h, eflour450 (eBioscience, 65-0842-85) at 5 mM (high concentration) was used to label peptide-pulsed cells at room temperature in the dark. Non-peptide-pulsed cells were labelled with a low concentration of eflour450 at 0.5 mM. After being rinsed three times with PBS, 4 × 106 labelled and peptide-pulsed cells and an equal number of labelled non-peptide-pulsed cells were adoptively transferred by tail vein injections into mice that had previously been immunized. Six hours later, the percentage of labelled cells in spleens was detected with LSRFortessa flow cytometry (BD) and analyzed by FlowJo (TreeStar). The following formula calculated the specific cell lysis: Specific cell lysis ability% = (1-(percentage of cells incubated with peptide/percentage of cells incubated without peptide)) x100%.
7
Assessing Antigen-Specific Cell Lysis
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Single suspensions of splenocytes from naïve syngeneic mice were diluted to 1.5*108/ml by RPMI1640 with 10% FBS and 2% Penicillin and Streptomycin, respectively, pulsed with or without 5 μg/ml peptides as mentioned above at 37°C. After 4 hours, a higher concentration of eflour450 (eBioscience, 65–0842-85) at 5 mM was used to label pulsed peptide cells. Cells without peptide-pulsed were labeled with a low concentration of eflour450 at 0.5 mM at room temperature in the dark. After being rinsed by PBS three times, 4*106 of labeled and peptides-pulsed cells and another equal number of labeled cells without peptide-pulsed were adoptive transferred by tail vein injections into mice previously immunized with different vaccines, respectively. Six hours later, the percentage of labeled cells was detected with LSRFortessa flow cytometry (BD) and analyzed by FlowJo (TreeStar). The following formula calculated the specific cell lysis: Specific cell lysis ability = (1-(percentage of cells incubated with peptide/percentage of cells incubated without peptide)) *100%.
8
Quantifying DR5 Expression in HCC-38 Cells
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HCC-38 cells were seeded onto 60 mm plates and treated with L-AA (50, 100, and 200 μM) for 24 h. The cells were treated with trypsin-EDTA and centrifuged at 1000 rpm for 5 min. After washing with PBS, the cells were incubated with anti-death receptor 5 (DR5) primary antibody (Abcam, Cambridge, MA, USA, 1:100) in blocking buffer (2% FBS in PBS) for 30 min on ice. The cells were washed and stained with anti-Alexa Fluor-488 secondary antibody (Invitrogen, Carlsbad, CA, 1:1000) or rabbit IgG monoclonal isotype control (Abcam, Cambridge, MA, USA) in the dark for 30 min on ice. DR5-positive cells were detected using LSRFortessa flow cytometry (BD Bioscience, San Jose, CA, USA).
9
HMC-FMX Cytotoxicity Assay in 22Rv1 Cells
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After culturing 22Rv1 cells overnight at 37 °C, the cells were treated with HMC-FMX at 8.0 μg/mL [HMC] and incubated at various time points: 0.5, 3, or 6 hours. Media was removed from the wells and rinsed twice with PBS. Cells were then fixed with 4% PFA at RT for 10 min and stained with DAPI for 15 min at RT. The cells were imaged using a fluorescence microscope.
For flow cytometric study, 22Rv1 cells were cultured overnight at 37 °C, followed by treatment with HMC-FMX at 8.0 μg/mL [HMC]. After incubating at various time points: 0.5, 3, or 6 hours, the medium was removed, and the cells were harvested by trypsinization. Then, cells were rinsed twice with PBS and fixed with ice-cold 4% PFA for 15 min. Cells were resuspended in FACS buffer (1% FBS and 0.05% NaN3 in PBS) and analyzed by LSR Fortessa flow cytometry (BD Biosciences, San Jose, CA) to record the fluorescence histograms of HMC dyes.
For flow cytometric study, 22Rv1 cells were cultured overnight at 37 °C, followed by treatment with HMC-FMX at 8.0 μg/mL [HMC]. After incubating at various time points: 0.5, 3, or 6 hours, the medium was removed, and the cells were harvested by trypsinization. Then, cells were rinsed twice with PBS and fixed with ice-cold 4% PFA for 15 min. Cells were resuspended in FACS buffer (1% FBS and 0.05% NaN3 in PBS) and analyzed by LSR Fortessa flow cytometry (BD Biosciences, San Jose, CA) to record the fluorescence histograms of HMC dyes.
10
Ovalbumin-specific B cell analysis
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Draining lymph nodes (dLN; inguinal) were harvested 7 days after each immunization. Cells were stained with W614A-3S coupled with biotinylated ovalbumin (Ova) protein (Covalab) for 30 min at room temperature before cell surface antigen staining with a standard method after receptor Fc blocking with CD16/CD32 (clone 2.4G2; BD Biosciences, San Jose, CA, USA), and the following anti-mouse Abs: CD3e (clone 145-2C11; eBioscience, San Diego, CA, USA), CD45R/B220 (clone RA3-6B2), CD19 (clone 1D3), IgG1 (clone A85-1), IgD (clone 11-26c.2a), T- and B-cell activation antigen (clone GL7), and streptavidin (BD Biosciences). For cell analysis, dead cells were excluded by using the LIVE/DEAD fixable kit (Molecular Probes, Eugene, OR, USA). Cells were analyzed by BD LSRFortessa flow cytometry or isolated by BD FACSAria II sorter. A pool of five mice per condition at W3 and W5 was used for W614A-3S-specific B cell isolations (BioMark Dynamic array). A pool of 25 mice per condition at W11 was used for W614A-3S-specific IgG1+ GC (GL7+IgDLow) and NGC (GL7-IgD+) B cell isolations [chromium single cell V(D)J assay].
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