Zymosan (0.1 mg) was injected i.p. into lipin-1mKO and littermate control mice and allowed to incubate for 6 d. Mice were then sacrificed, and the peritoneal lavage was collected in FACS wash buffer (1% BSA, 1 mM EDTA, and 0.1% sodium azide in PBS). A total of 500,000 isolated peritoneal cells were blocked with CD16/CD32 (1:200) (14-0161-86; eBioscience) for 20 min. Cells were incubated with PECy7-conjugated CD11b (1:4000) (25-0112-81, clone M1/70; eBioscience), AF700-conjugated anti-CD45.2 (1:2000) (109821, clone 104; BioLegend), FITC-conjugated anti-Ly6G (1:800) (551460, clone 1A8; BD Biosciences), PECy5-conjugated anti-F4/80 (1:400) (15-4801-80, clone BM8; Invitrogen), and PE-conjugated anti-MerTK Ab (1:500) (151506; BioLegend) for 30 min in the dark. Cells were spun at 400 × g for 5 min and resuspended in FACS fix (1% paraformaldehyde in FACS wash buffer). Appropriate F Minus One Controls were used to identify positive staining. Compensation controls (Comp Bead, 01-2222-42; Invitrogen) were used to exclude spectral overlap. Flow cytometry was performed using Quanteon flow cytometer (Acea Biosciences). Data analysis was performed using FCS express (Denovo Software) and NovoExpress (Acea Biosciences).
Quanteon flow cytometer
Manufactured by Agilent Technologies
Sourced in United States
The Quanteon flow cytometer is a laboratory instrument designed for the analysis and sorting of cells and particles in liquid samples. It utilizes advanced technology to detect and measure various characteristics of individual cells or particles as they flow through a laser-based detection system. The core function of the Quanteon is to provide accurate and reliable data on the properties of the analyzed samples, enabling researchers and scientists to study cellular populations and dynamics.
Automatically generated - may contain errors
Lab products found in correlation
Cd16 cd32, by Thermo Fisher Scientific (2 mentions)
Fcs express, by De Novo Software (2 mentions)
Novoexpress, by Agilent Technologies (2 mentions)
Comp bead, by Thermo Fisher Scientific (2 mentions)
St511, by Beyotime (1 mentions)
Legendplex human inflammation panel 1, by BioLegend (1 mentions)
Meso quickplex sq 120, by Mesoscale (1 mentions)
Alexa fluor antibody labeling kit, by Thermo Fisher Scientific (1 mentions)
Ma900 cell sorter, by Sony (1 mentions)
8 protocols using quanteon flow cytometer
1
Quantification of Peritoneal Immune Cells
2
Macrophage Subpopulation Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
FITC anti-CD14+ cell antibody (Biolegend, 367,116), PerCP-Cy5.5-anti-CD9+ cell antibody (Biolegend, 312,110), PE/Cy7 anti-CSF1R antibody (Biolegend, 347,308) and APC-TREM2+ cell antibody (R&D Systems, FAB17291A) were used to analyze the ratio of CD9+TREM2+ cells of macrophages in PBMCs of T3-treated samples and DMSO-treated controls. The Quanteon flow cytometer (ACEA Biosciences) was used to perform flow cytometry.
3
Apoptosis Assay in 2D and 3D Cell Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells in 2D culture were planted in 6-well culture plates at a density of 3 × 105 cells/well. After 24 h of incubation, cells were treated with drugs alone or in combination, with the optimal concentration according to the MTT assay results. After the drug treatment for 24 h, the cells were digested with EDTA-free trypsin solution, harvested, and then resuspended in 500 µL Binding Buffer (1×) with 5 µL Annexin V-FITC and 5 µL PI. After incubation for 15 min at room temperature in the dark, the samples were analyzed using a Quanteon flow cytometer (ACEA, USA). Apoptosis rate = early apoptosis rate + late apoptosis rate. The experiment was repeated three times.
Cells in 3D culture were seeded in 24-well plates at a density of 25,000 cells/50µL Matrigel. After 7 days of incubation, drug intervention was performed. After the drug treatment for 72 h, the supernatant was discarded and Cell Recovery Solution was used to dissolve Matrigel to collect 3D cell clusters, and EDTA-free trypsin was applied to digest the cell clusters into single cell suspension. The Annexin V-FITC and PI staining assay in 3D culture was the same as described in 2D culture. The experiment was repeated three times.
Cells in 3D culture were seeded in 24-well plates at a density of 25,000 cells/50µL Matrigel. After 7 days of incubation, drug intervention was performed. After the drug treatment for 72 h, the supernatant was discarded and Cell Recovery Solution was used to dissolve Matrigel to collect 3D cell clusters, and EDTA-free trypsin was applied to digest the cell clusters into single cell suspension. The Annexin V-FITC and PI staining assay in 3D culture was the same as described in 2D culture. The experiment was repeated three times.
4
Quantification of Peritoneal Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Zymosan (0.1 mg) was injected i.p. into lipin-1mKO and littermate control mice and allowed to incubate for 6 d. Mice were then sacrificed, and the peritoneal lavage was collected in FACS wash buffer (1% BSA, 1 mM EDTA, and 0.1% sodium azide in PBS). A total of 500,000 isolated peritoneal cells were blocked with CD16/CD32 (1:200) (14-0161-86; eBioscience) for 20 min. Cells were incubated with PECy7-conjugated CD11b (1:4000) (25-0112-81, clone M1/70; eBioscience), AF700-conjugated anti-CD45.2 (1:2000) (109821, clone 104; BioLegend), FITC-conjugated anti-Ly6G (1:800) (551460, clone 1A8; BD Biosciences), PECy5-conjugated anti-F4/80 (1:400) (15-4801-80, clone BM8; Invitrogen), and PE-conjugated anti-MerTK Ab (1:500) (151506; BioLegend) for 30 min in the dark. Cells were spun at 400 × g for 5 min and resuspended in FACS fix (1% paraformaldehyde in FACS wash buffer). Appropriate F Minus One Controls were used to identify positive staining. Compensation controls (Comp Bead, 01-2222-42; Invitrogen) were used to exclude spectral overlap. Flow cytometry was performed using Quanteon flow cytometer (Acea Biosciences). Data analysis was performed using FCS express (Denovo Software) and NovoExpress (Acea Biosciences).
5
DNA Content Analysis by FACS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA content was analyzed with fluorescence-activated cell sorting (FACS). For the FACS analysis, the cells were first fixed with cold 70% ethanol and kept on ice for >30 min before pelleting at 12,000 rpm. Cells were then resuspended in 50 mM sodium citrate with 10 µg/mL RNase A for over 3 h. DNA was then stained with 2 μg/mL propidium iodide (PI) (Beyotime Biotechnology, ST511) before brief sonication. At least 20,000 cells were acquired per sample on a Quanteon flow cytometer (ACEA Biosciences) for PI detection (FL2-A). DNA content is shown on a linear scale after gating for single cells in FlowJo (version 10) software.
6
Annexin V-FITC/PI Apoptosis Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were harvested by centrifugation at 1500 rpm for 5 min and washed with PBS for 3 times, and apoptosis was measured using the Annexin V-FITC/PI Apoptosis Assay Kit. Stained cells were detected using a Quanteon flow cytometer (Agilent, California, USA).
7
Quantification of Cytokine Profiles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cytokine concentrations in ALO supernatants were quantified using a LEGENDplex Human Inflammation Panel 1 (BioLegend) kit and read on a Quanteon flow cytometer (Agilent). Data were analyzed using LEGENDplex v8.0 software. Cytokine levels in gNVU perfused media were quantified using the MSD V-PLEX Proinflammatory Panel Human Kit (Meso Scale Discovery) and MESO QuickPlex SQ 120 (Meso Scale Discovery) reader.
8
Fluorochrome Labeling and Cell Sorting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Monoclonal antibodies were first labeled with Fluorochrome using Alexa Fluor™ Antibody Labeling Kits (ThermoFisher Scientific, Waltham, MA, cat # A20181 or A20186) and then 1 µg was used to label 106 cells in 100 µl 1X PBS, 0.1% glucose and 2% FBS (cell wash buffer) for 30 min at 4 oC. Following labeling, cells were washed twice with cell wash buffer and resuspended in 100 µl cell wash buffer. Flow cytometric analyses were performed in a Quanteon Flow Cytometer (Agilent, Santa Clara, CA). Flow gating strategies were illustrated in Supplementary Fig. 2 . Data was analyzed with FlowJo Software v10.8. Stable transfected cells stained with FM5148-Alexa Fluor 488 were sorted on a MA900 cell sorter (Sony Biotechnology, San Jose, CA). Sorting strategies were demonstrated in Supplementary Fig. 3a and b . Cells with high expression of HLA-DQα (FM5148-Alexa Fluor 488) were sorted and plated in RPMI-1640 + 10% FBS with the corresponding selection antibiotics.
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!