To perform flow cytometry, cells were acquired from the alveolar compartment of LPS-exposed or unchallenged mice by centrifugation of BALF samples (300g for 5 min), or from total lung tissue by enzymatic digestion of intact, perfused lungs as described (13 (link)). Cell suspensions were then subjected to red blood cell lysis (BioLegend, 10 min incubation at RT), live/dead cell staining (BioLegend, Zombie Violet, 10 min incubation at RT) and Fc receptor blocking (BD Pharmingen, Purified Rat Anti-Mouse CD16/CD32, 1:50, diluted in PBS supplemented with 10% mouse serum and 1% BSA, 15 min incubation on ice) (13 (link)). Cells were then stained for 1 h on ice using the following surface marker antibodies: anti-CD45 (clone 30-F11; BV605; 1:100 dilution; BioLegend), anti-CD11b (clone M1/70; AF488; 1:200; BioLegend), anti-CD11c (clone N418; allophycocyanin; 1:200, BioLegend), anti-F4/80 (clone BM8; PE; 1:50; BioLegend), anti-CD206/MR (clone C068C2; PE-Cy7; 1:100; BioLegend). PBS supplemented with 1% BSA was used for dilution of antibodies and a subsequent wash. After antibody staining, cells were filtered through a 40-μm mesh and then analyzed using a Fortessa 20× instrument (BD Biosciences). Gates identifying cells positive for each marker were defined using fluorescence-minus-one controls in which each antibody in turn was excluded.
Zombie violet
Manufactured by BD
Zombie Violet is a laboratory equipment designed for specialized applications. It serves as a core functional component within the broader spectrum of lab equipment utilized for various research and testing purposes. The specific details and intended use of this product are not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Purified rat anti mouse cd16 cd32, by BD (1 mentions)
Pharm lyse solution, by BD (1 mentions)
Anti mouse cd16 cd32 antibody, by BD (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Anti f4 80, by Thermo Fisher Scientific (1 mentions)
Anti cd45, by BioLegend (1 mentions)
Anti ly6g ly6c, by BioLegend (1 mentions)
Anti cd11c, by BioLegend (1 mentions)
Anti cd11b, by BioLegend (1 mentions)
F4 80, by BioLegend (1 mentions)
3 protocols using zombie violet
1
Flow Cytometry of Lung Cells
2
Optimized Flow Cytometry Protocols for Diverse Cell Types
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FCM analyses and cell sorting for blood30 (link) and tissue64 (link) cells were performed as previously described. lood samples were mixed with PBS, and red blood cells were lysed using the BD Pharm Lyse solution (BD Biosciences, Franklin Lakes, NJ, USA). Zombie Violet (BD Biosciences) treatment was performed to eliminate dead cells. The samples were then incubated on ice with an anti-mouse CD16/CD32 antibody (BD Biosciences, 1:100) to block Fc receptors. The cells were incubated with the fluorescence-labelled antibodies listed in key resources table on ice for 30 min. After centrifugation, the pellets were resuspended in 250 μl of PBS containing 2% fetal bovine serum. Flow cytometry was performed using a Gallios flow cytometer (Beckman Coulter). The data were analyzed using FlowJo software (version 7.6.5; Treestar, Ashland, OR, USA). For FCM analysis of brain and lung tissues, tissues were dissociated into single cells using their respective dissociation kit following the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany), and the dissociated cells were processed as blood samples using the same method. Total RNA was collected from sorted cells and fixed overnight with a cell cover (Anacyte Laboratories, Hamburg, Germany).14 (link)
3
Isolating and Characterizing Kidney Macrophages
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The left kidney from each mouse was isolated and transferred to PBS on ice. Kidneys were weighed, cut into small pieces, and digested in a mix of enzymes. Briefly, collagenase type XI (125 U/mL), collagenase type IS (450 U/mL), and hyaluronidase IV-S (60 U/mL) were used for digestion at 37 °C for 20 minutes, with regular agitation. The digested tissue was then passed through a 70 μm sterile cell strainer (Falcon; BD Biosciences, San Jose, CA) to yield a single-cell suspension. Cells were washed and resuspended in fluorescence-activated cell sorting buffer, counted, stained, and collected using multicolor flow cytometry (BD LSR II flow cytometer with DIVA software, BD Biosciences). The macrophage population was defined as CD45+F4/80+CD11b+ cells and further characterized for M1 proinflammatory macrophage (CD11c+) as previously described.41 (link) Following antibodies were used in the panel: anti-CD45, anti-Ly-6G/Ly-6C, anti-CD11b, anti-CD11c (all from BioLegend), and anti-F4/80 (eBioscience). Dead cells were eliminated from analysis using Zombie Violet (BD Biosciences). For each experiment, fluorescence minus one control for each fluorophore was performed to establish gates. In selected experiments, accuracy of the fluorescence minus one gating strategy was confirmed using isotype controls. Data were analyzed by Flow Jo v.10 (Ashland, OR).
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