Tumorsphere-derived cells were stained with the Aldefluor kit (Stem Cell Technologies, Grenoble, France) as reported [51 (link)]. To measure intracellular ROS content, cells were stained with 2′, 7′-dihydrochlorofluorescein diacetate (DHCF-DA, Sigma-Aldrich) as described [9 (link)]. The results were expressed as percentages of positive cells and as mean fluorescence intensity (MFI). For the analysis of immune infiltrates, lungs and tumors from vaccinated mice were finely minced with scissors and then digested by incubation with 1 mg/mL of collagenase IV (Sigma Aldrich) in RPMI-1640 (Life Technologies) at 37 °C for 1 h in an orbital shaker. After washing in PBS, cell suspensions were incubated with a buffer for erythrocyte lysis (155 mM NH4Cl, 15.8 mM Na2CO3, 1 mM EDTA, pH 7.3) for 10 min at room temperature, then passed through a 40 µm-pore cell strainer, centrifuged at 1800 rpm for 10 min and suspended in PBS. The cells were treated with an Fc receptor (FcR) blocker (CD16/CD32; Becton Dickinson) for 5 min at 4 °C and then stained for 30 min at 4 °C with the following Abs: anti-mouse CD45 VioGreen, anti-mouse CD3 FITC, anti-mouse CD4 APC-Vio770, anti-mouse CD8 VioBlue, and anti-mouse CD49b PE (all from Miltenyi Biotec, Bologna, Italy). All samples were acquired on a BD FACSVerse and analyzed with the BD FACSuite™ software (Becton Dickinson, Milan, Italy).
Dhcf da
Manufactured by Merck Group
DHCF-DA is a fluorescent dye used in laboratory settings to measure cellular health and viability. It is a cell-permeant dye that becomes fluorescent upon oxidation, indicating the presence of reactive oxygen species within the cell.
Automatically generated - may contain errors
Lab products found in correlation
Polarstar optima microplate reader, by BMG Labtech (2 mentions)
Facsverse, by BD (1 mentions)
Anti mouse cd3 fitc, by Miltenyi Biotec (1 mentions)
Facsuite software, by BD (1 mentions)
Anti mouse cd45 viogreen, by Miltenyi Biotec (1 mentions)
Anti mouse cd49b pe, by Miltenyi Biotec (1 mentions)
Anti mouse cd4 apc vio770, by Miltenyi Biotec (1 mentions)
Anti mouse cd8 vioblue, by Miltenyi Biotec (1 mentions)
Cd16 cd32, by BD (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Collagenase 4, by Merck Group (1 mentions)
Aldefluor kit, by STEMCELL (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
4 protocols using dhcf da
1
Quantifying Tumor-Infiltrating Immune Cells
2
Intracellular ROS Measurement in M. catarrhalis Infection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
1x106 cells in 200 µl medium were incubated in the dark with 10 µg/ml of 2′,7′Dihydrodichlorofluorescein diacetate (DHCF‐DA) (Sigma) for 30 min prior to infection with M. catarrhalis for 1 h at multiplicity of infection (MOI) 100. Thereafter cells were centrifuged, resuspended in PBS, and fluorescence was analysed by flow cytometry.
3
Arsenic-Induced Oxidative Stress Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded at a density of 3 Â 10 4 cells per cm 2 in 12-well plates. Nine days after seeding, the monolayers were exposed for 2, 4 and 24 h to As(III) (1 and 3 mg L À1 ) and As(V) (5 and 8 mg L À1 ) prepared in MEMc.
After treatment, the cells were washed with phosphate-buffered saline (PBS, Hyclone) and they were incubated for 30 min at 37 1C with a solution of 200 mL of 100 mM 2 0 ,7 0 -dihydrochlorofluorescein diacetate (DHCF-DA, Sigma) prepared in PBS. After that time the cells were lysed using 300 mL of Triton X-100 (0.1% m/v in PBS) (Merck). After sonication (10 min, 4 1C) and centrifugation (11 000 rpm, 3 min), the cell lysate was transferred to a 96-well plate and the fluorescence was determined (excitation l = 488 nm; emission l = 530 nm) using a PolarSTAR OPTIMA microplate reader (BMG-Labtech, Cary, NC, USA). The fluorescence values obtained were normalized per mg of protein.
After treatment, the cells were washed with phosphate-buffered saline (PBS, Hyclone) and they were incubated for 30 min at 37 1C with a solution of 200 mL of 100 mM 2 0 ,7 0 -dihydrochlorofluorescein diacetate (DHCF-DA, Sigma) prepared in PBS. After that time the cells were lysed using 300 mL of Triton X-100 (0.1% m/v in PBS) (Merck). After sonication (10 min, 4 1C) and centrifugation (11 000 rpm, 3 min), the cell lysate was transferred to a 96-well plate and the fluorescence was determined (excitation l = 488 nm; emission l = 530 nm) using a PolarSTAR OPTIMA microplate reader (BMG-Labtech, Cary, NC, USA). The fluorescence values obtained were normalized per mg of protein.
4
Fluoride Exposure and Oxidative Stress
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were seeded in 24-well plates at a density of 2.6 × 10 4 cells/cm 2 . After reaching confluence, cells were exposed over 4, 24 and 48 h to different concentrations of fluoride (2.5, 5, 10, 20, 50 and 65 mg/L, equivalent to 0.13, 0.26, 0.53, 1.1, 2.6 and 3.7 mM NaF, respectively) prepared in supplemented MEM. Cells treated with 2 mM H 2 O 2 (Prolabo) were used as positive controls.
Following the treatments, the medium was removed and the cells were washed with PBS.
Then 100 μL of 2',7'-dichlorofluorescein diacetate 100 µM (DHCF-DA, Sigma) prepared in PBS were added, followed by incubation at 37ºC for 30 min. After this time the medium was removed and the cells were washed with PBS and lysed using 150 µL of a solution of Triton X-100 (1% w/v in PBS). After sonication for 10 min at 4ºC and centrifugation at 11000 rpm for 3 min, 100 µL of cell lysate was transferred to a 96-well plate and the fluorescence was determined (λ excitation = 488 nm; λ emission = 530 nm) using a PolarSTAR OPTIMA microplate reader (BMG-Labtech, Germany). The fluorescence values obtained were standardized per mg of protein, quantified by the Bradford method (Bio-Rad Protein Assay, Bio-Rad, USA).
The results (arbitraty units of fluorescence/mg protein) were expressed as percentages with respect to the cells not treated with fluoride or H 2 O 2 .
Following the treatments, the medium was removed and the cells were washed with PBS.
Then 100 μL of 2',7'-dichlorofluorescein diacetate 100 µM (DHCF-DA, Sigma) prepared in PBS were added, followed by incubation at 37ºC for 30 min. After this time the medium was removed and the cells were washed with PBS and lysed using 150 µL of a solution of Triton X-100 (1% w/v in PBS). After sonication for 10 min at 4ºC and centrifugation at 11000 rpm for 3 min, 100 µL of cell lysate was transferred to a 96-well plate and the fluorescence was determined (λ excitation = 488 nm; λ emission = 530 nm) using a PolarSTAR OPTIMA microplate reader (BMG-Labtech, Germany). The fluorescence values obtained were standardized per mg of protein, quantified by the Bradford method (Bio-Rad Protein Assay, Bio-Rad, USA).
The results (arbitraty units of fluorescence/mg protein) were expressed as percentages with respect to the cells not treated with fluoride or H 2 O 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!