To determine the absolute numbers of Gr-1+ CD11b+ granulocytes in the lungs from infected animals, flow cytometric analysis of single-cell suspensions was performed. After blocking of nonspecific binding by adding an anti-FcγRIII/II antibody (BioLegend, Amsterdam, The Netherlands) and a cocktail of mouse, rat, and hamster serum, cells were incubated with optimal amounts of the following specific antibodies (all BD Biosciences, Heidelberg, Germany): anti-CD8-V450, anti-CD4-V500, anti-CD11c-FITC, anti-CD11b-PE, anti-Ly-6G-PerCP-Cy5.5, anti-CD90.2-PE-Cy7, anti-Gr-1-APC, and anti-MHCII(IA/IE)-APC-e780. Measurement was performed on a FACSCanto™ II (BD Bioscience) and the FCS files were analyzed using the FCS Express 7 Flow Cytometry software (DeNovo™ Software, Pasadena, CA, USA). Absolute cell numbers were calculated as follows: (total cell count lung/100) × % of Gr-1+ CD11b+ cells (of analyzed flow cytometric leukocytes) = absolute cell numbers.
Fcs express 7 flow cytometry software
Manufactured by De Novo Software
Sourced in United States
FCS Express 7 Flow Cytometry software is a powerful data analysis tool designed for the interpretation of flow cytometry data. The software provides a comprehensive suite of analysis tools and visualization options to assist researchers in the assessment and interpretation of their flow cytometry experiments.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (1 mentions)
Anti cd11b pe, by BD (1 mentions)
Anti cd4 v500, by BD (1 mentions)
Anti cd11c fitc, by BD (1 mentions)
Anti cd8 v450, by BD (1 mentions)
Anti cd90.2 pe cy7, by BD (1 mentions)
Anti fcγriii 2 antibody, by BioLegend (1 mentions)
Facsaria 2 cell sorter, by BD (1 mentions)
Tryple express, by Thermo Fisher Scientific (1 mentions)
Lsr fortessa cytometer, by BD (1 mentions)
3 protocols using fcs express 7 flow cytometry software
1
Quantification of Gr-1+ CD11b+ Granulocytes in Infected Lungs
2
FACS Sorting of ACE-tRNA-Expressing Cells
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For FACS experiment, cells were washed with Dulbecco’s phosphate-buffered saline (DPBS) (Gibco; no. 14190-144) twice, dissociated with TrypLE Express (Gibco; no. 12604-013), pelleted at 1,000 rpm (∼500 × g) for 5 min, resuspended with DPBS with calcium and magnesium (Gibco; no. 14040-133), and strained with 40 μm Cell Strainer. Cells were then sorted using the BD FACSAria II cell sorter (BD Biosciences), and data were analyzed by using FCS Express 7 flow cytometry software (De Novo Software, Pasadena, CA, USA).
In order to sort ACE-tRNA-expressing cells from ACE-tRNA-not-expressing cells, HBE cells were transfected with the plasmid-containing ACE-tRNA and mNG fluorescent protein in one plasmid. Approximately 36–48 h after transfection, cells were prepared as described above for FACS experiment. Empty-vector-expressing cells were used to determine the gate for mNG-negative and positive cells. Each population of mNG-negative and positive cells was then sorted into a 15-mL conical tube using the BD FACSAria II cell sorter. Total RNA was isolated from sorted cells and used for reverse transcriptase and quantitative PCR (RT-qPCR) as described below.
In order to sort ACE-tRNA-expressing cells from ACE-tRNA-not-expressing cells, HBE cells were transfected with the plasmid-containing ACE-tRNA and mNG fluorescent protein in one plasmid. Approximately 36–48 h after transfection, cells were prepared as described above for FACS experiment. Empty-vector-expressing cells were used to determine the gate for mNG-negative and positive cells. Each population of mNG-negative and positive cells was then sorted into a 15-mL conical tube using the BD FACSAria II cell sorter. Total RNA was isolated from sorted cells and used for reverse transcriptase and quantitative PCR (RT-qPCR) as described below.
3
Evaluating CSC Population Dynamics
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The direct effect of the drugs on the CSC population was studied by flow cytometry in ALDH1A-tdTomato CSC models. For each cell line, tdTomato+ and tdTomato- cell subpopulations corresponding to the CSC and non-CSC cells, respectively, were maintained at a 1:1 ratio. Cells were seeded on 6-well plates at a density of 200,000 cells per well. After 24 h, the cells were incubated with the selected treatments (individual and in combination) for 72 h. Thereafter, the medium was removed, and the cultures were refed with complete medium to allow cellular recovery in the absence of drug/s for an additional 48 h. Changes in the amount of tdTomato+ were evaluated using the BD LSRFortessa™ Cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and subsequently analyzed using FCS express 7 Flow cytometry software (De novo, Pasadena, CA, USA) to calculate the increase or decrease in the tdTomato+ population.
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