The generation of reactive oxygen species (ROS) inside the amastigotes of Leishmania braziliensis via mechanical action was monitored using 2′7′dihydrofluorescein (H2DCF-DA) as a fluorescent probe. The cell concentration of the parasites was adjusted to 1 × 107 cells/mL in M199 or RPMI-1640 medium without phenol red at pH 5.5 supplemented with 10% fetal bovine serum (FBS). After washing the cells via centrifugation with phosphate-buffered saline (PBS), they were resuspended in 100 μL of H2DCF-DA probe (0.2 μg/mL Hanks’s solution) and seeded into 96-well dark microplates, which were incubated for 1 h at 32 °C under protection from light. Then, dichlorofluorescein, which was derived from the oxidation of H2DCFDA, was quantified based on ROS levels using a flow cytometer (LSR Fortesa, BD Biosciences, USA) [10 (link)].
Lsrfortesa
Manufactured by BD
Sourced in United States
The BD LSRFortesa is a flow cytometer designed for advanced multicolor analysis. It features a compact design and offers high-performance capabilities for a range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Facsdiva software, by BD (3 mentions)
Fixation permeabilisation buffer, by Thermo Fisher Scientific (1 mentions)
Flowlogic software, by Inivai Technologies (1 mentions)
Varioskan flash, by Thermo Fisher Scientific (1 mentions)
Anti lacc1 e7 clone, by Santa Cruz Biotechnology (1 mentions)
Lsrii apparatus, by BD (1 mentions)
Foxp3 transcription staining buffer set, by Thermo Fisher Scientific (1 mentions)
Facsaria instrument, by BD (1 mentions)
Monensin, by Thermo Fisher Scientific (1 mentions)
Human cd3 apc cy7, by BioLegend (1 mentions)
Cd19 bv650 hib19, by BioLegend (1 mentions)
Cd7 pe, by BD (1 mentions)
Golgiplug, by BD (1 mentions)
Cd56 apc, by BD (1 mentions)
Mq1 17h12, by BioLegend (1 mentions)
Guava easy cyte cytometer, by Merck Group (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Facscanto, by BD (1 mentions)
17 protocols using lsrfortesa
1
Monitoring ROS generation in Leishmania
2
Multiparametric Flow Cytometry Analysis of Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Thymic glands, spleens and inguinal lymph nodes (ILNs) were harvested from Hpa-tg and WT mice 7-days post CIA injection (n = 7). Single cell suspensions from these organs were prepared as described earlier31 (link) and stained with surface markers: CD4, CD8, CD25, CXCR5, PD-1, CD19, B220, Ly6G, CD11c, PDCA-1, CXCR6 and CD11b (Supplementary Table S2 ). The cells were then fixed and permeabilised with Fixation Permeabilisation buffer (eBioscience) for staining of intracellular markers: Foxp3, Helios, IFN-γ, Ki-67 and IL-17a (Supplementary Table S2 ). The samples were analysed on LSRFortesa (BD) using DivaDacker software (BD). One million events were counted for each sample and the fluorescence minus one and single stained controls were used for gating strategies as described earlier31 (link). The FCS files were analysed on FlowLogic software (Inivai Technologies).
3
ROS Generation in Leishmania Amastigotes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The generation of reactive oxygen species (ROS) was assessed by flow cytometry in axenic amastigotes of L. pifanoi and mechanical released amastigotes of L. braziliensis, using the 2′7′-dihydrofluorescein (H2DCF-DA) probe (Carvalho et al., 2011 (link)). The dichlorofluorescein resulting from the oxidation of H2DCF-DA by ROS in L. pifanoi was quantified in the microplate reader (Varioskan Flash, Thermo Fisher Scientific, USA) at a wavelength of 495 nm excitation and 527 nm emission. In L. braziliensis, the amount of dichlorofluorescein was measured in a flow cytometer (LSR Fortesa, BD Biosciences, USA), according to the median fluorescence intensity (MFI). Antimycin (0.3 μg/mL) was used as control in L. pifanoi, while rotenone (50 μg/mL) was used as control in L. braziliensis experiments.
4
LACC1 Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
U937 cells were fixed with eBioscience Fixation/Permeabilization Concentrate (00-5123-43; Invivogen) in combination with the 10× Permeabilization buffer (00–8333-56) following the manufacturer’s instructions. Cells were then stained with primary antibodies (anti-LACC1 [E7 clone; Santa Cruz], anti-LACC1 [HPA040150; Sigma-Aldrich], or anti-HA [901524; Biolegend]) for 1 h at room temperature. Secondary antibodies used were anti-rabbit or anti-mouse coupled with Alexa Fluor 747 for 30 min. Cells were then acquired using a LSRFortesa (BD Biosciences), and data were analyzed using FlowJo (version 10.2)
5
Lymphocyte Profiling by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Phenotyping of lymphocyte populations was performed by flow cytometry after preparation of single cell suspensions from lymphoid organs and staining using antibodies listed in Table S6. Single cell suspension of thymus, spleen and lymph nodes were prepared in FACS buffer (2% FBS, 1 mM EDTA, 1% penicillin-streptomycin, in PBS) by tissue homogenization with a syringe plunger against a 40 mm cell strainer. For preparation of cell suspensions from bone marrow, femur, tibia and pelvis were flushed with FACS buffer using a 10 mL syringe and a 26 gauge-needle and then passed through 40 mm cell strainer to obtain single cell suspensions. To achieve red blood cell lysis, the cell suspensions were treated with ACK lysis buffer (0.15M NH4Cl, 10mM KHCO3, 0.1mM EDTA in H2O, pH 7.2-7.4), washed and resuspended in FACS buffer. For intracellular staining the cells were first stained with a fixable viability dye prior to surface antibody staining. After cell surface staining the cells were fixed and permeabilized using the FoxP3/Transcription staining buffer set (eBioscience) according to manufacturer's protocol followed by intracellular antibody staining. Data were collected on an LSRFortesa and/or LSRII apparatus (BD Biosciences) and were analyzed with FlowJo software version 10; cell sorting was done using a FACSAria instrument (BD Biosciences).
6
Multiparametric Flow Cytometry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single cell suspension was processed and stained according to standard protocols. In brief, cell number was adjusted to a concentration of 1–5 × 106 cells/mL in ice-cold FACS Buffer (PBS, 5% FBS, 5 mM EDTA). Then, cells were stained with labeled antibodies for 20 min at 4 °C, washed with FACS buffer and measured. The following antibodies were used: fluorochrome-conjugated monoclonal antibodies against human CD3-APC Cy7 (BioLegend, San Diego, CA, USA), CD5-BV421 (UCHT2, BioLegend, San Diego, CA, USA), CD7-PE (BD Pharmingen, Franklin lakes, NJ, USA), CD19-BV650 (HIB19, BioLegend, San Diego, CA, USA), CD56-APC (BD Pharmingen, Franklin lakes, NJ, USA), CD107a (Invitrogen, Waltham, MA, USA). For intracellular staining, cells were fixed and permeabilized with protein transport inhibitor Golgi Plug (BD, Franklin lakes, NJ, USA) and Monensin (eBioscience, San Diego, CA, USA). Cells were analyzed on a LSR Fortesa (BD Biosciences) using FACSDiva software v.9.0 (Franklin lakes, NJ, USA) Data were analyzed using FlowJo software v.10.7.1 (Tree Star, Ashland, OR, USA).
7
Multiparametric Flow Cytometry Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To assess surface expression of CD11a and CD3ε on Jurkat cells, 0.25 × 106 cells/sample were washed and blocked with 2% FBS/PBS for 30 min at 4°C and then stained with anti-CD3ε-Alexa Fluor 488 (BioLegend, #317310) and anti-CD11a (ThermoFisher, #MA11A10) conjugated in house with Alexa Fluor 647 (ThermoFisher, #A20006), at 10 μg/mL for 30 min at 4°C. Finally, cells were washed three times in 2% FBS/PBS, fixed in 2% PFA/PBS, analyzed by LSRFortesa (BD Biosciences) with BD FACSDiva software and plotted using FlowJo version 10.
Flow cytometric analysis of surface CD69 was carried out using FITC anti-CD69, clone FN50 (BioLegend, #310904) at 1 μg/ml for 30 min at 4°C. Samples were analyzed with Guava Easy Cyte Cytometer (Millipore) and plotted using FlowJo version 6.1.1.
IL-2 intracellular staining flow cytometry was carried out using APC-labeled anti-human IL-2, clone MQ1-17H12 (BioLegend, #500310), at 0.125 μg/5 × 105 cells. Samples were analyzed using a Becton Dickinson FACS CANTO II with BD FACSDiva 6.0 software.
For all experiments unstained cells and isotype controls were performed for background correction and gating.
Flow cytometric analysis of surface CD69 was carried out using FITC anti-CD69, clone FN50 (BioLegend, #310904) at 1 μg/ml for 30 min at 4°C. Samples were analyzed with Guava Easy Cyte Cytometer (Millipore) and plotted using FlowJo version 6.1.1.
IL-2 intracellular staining flow cytometry was carried out using APC-labeled anti-human IL-2, clone MQ1-17H12 (BioLegend, #500310), at 0.125 μg/5 × 105 cells. Samples were analyzed using a Becton Dickinson FACS CANTO II with BD FACSDiva 6.0 software.
For all experiments unstained cells and isotype controls were performed for background correction and gating.
8
Characterizing TILs and Splenocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TILs and splenocytes were stained ex vivo for cell surface expression of CD8, Vβ7, CD44, CD62L and PD-1 (Biolegend). After in vitro stimulation, TILs and splenocytes were stained for cell surface expression of CD8 and Vβ7, and intracellular accumulation of IFNγ and granzyme B (Biolegend). Intracellular staining of the cells was performed using the cytofix/cytoperm kit (BD Biosciences). Samples were acquired using either FACSCanto or LSR Fortesa operated by FACS Diva software (BD Biosciences). Data analysis was performed using FlowJo software (Tree Star).
9
Cell Cycle Analysis via EdU and PI
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For cell cycle analysis, about 1 × 106 cells were processed with the Click-iT EdU Flow Cytometry Assay Kits (Invitrogen) according to the manufacturer’s instructions. In brief, the cells were harvested after 2 h incubation with EdU and stained with Alexa Fluor 647 dye azide and propidium iodide. Cells were examined by fluorescence-activated cell sorting (FACS) using a flow cytometer (BD LSRFortesa).
10
Coumarin-6 Cellular Uptake Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NPcoumarin 6 was obtained with 1.3 µg/mL coumarin-6 as described above. SJSA-1 cells and 143B cells were seeded in 96-Well Ultra Low Attachment Microplate, and then treated with different coumarin-6 formulations. After an incubation for 30 min, the cells were washed, fixed in 4% paraformaldehyde, and stained with DAPI. Images were obtained using a fluorescent microscope. Fluorescence intensity was further determined using flow cytometry (BD LSRFortesa).
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!