Blood was collected via cardiac puncture then brain and spleen were sampled after transcardial perfusion with saline. Peripheral blood mononuclear cells and splenocytes were prepared by using red cell lysis buffer (eBioscience, San Diego, California). For brain immune cells, the forebrain was dissected from the cerebellum and suspended in RPMI-1640 medium (Corning, Pittsburgh, Pennsylvania). The suspension was digested with type IV collagenase (1 mg/ml, MP Biomedicals, Solon, Ohio) and DNase I (50 μg/ml, Roche Diagnostics, Indianapolis, Indiana) at 37°C for 45 min, then immune cells were isolated by 37–70% Percoll (GE Healthcare, Piscataway, New Jersey) density gradient centrifugation and collected from the interface. Single cell suspension was washed with staining buffer (PBS containing 0.1% NaN3 and 2% FCS) and stained with CD45 (30-F11, eBioscience), CD11b (M1/70, eBioscience), and Gr1 (IA8, BD Bioscience). Propidium iodide (PI, 2 μg/ml, Sigma) was used to exclude dead cells. Appropriate isotype control antibodies were applied to set quadrants for calculating the percentage of positive cells. Data were acquired on FACS Calibur (BD) and analyzed with FlowJo software (TreeStar, Ashland, Oregon).
Type 4 collagenase
Manufactured by MP Biomedicals
Type IV collagenase is an enzyme used in cell and tissue isolation and dissociation procedures. It selectively degrades type IV collagen, a structural component of basement membranes.
Automatically generated - may contain errors
Lab products found in correlation
Red cell lysis buffer, by Thermo Fisher Scientific (1 mentions)
Cd11b m1 70, by Thermo Fisher Scientific (1 mentions)
Cd45 30 f11, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Facscalibur, by BD (1 mentions)
Dnase 1, by Roche (1 mentions)
Rpmi 1640 medium, by Corning (1 mentions)
Gentlemacs c tube, by Miltenyi Biotec (1 mentions)
Stabilizing fixative, by BD (1 mentions)
Cd8 apc, by Thermo Fisher Scientific (1 mentions)
Cd3 percp cy5, by Thermo Fisher Scientific (1 mentions)
Cd11b alexafluor488, by Thermo Fisher Scientific (1 mentions)
Cd4 alexa fluor 488, by BioLegend (1 mentions)
Anti cd16 cd32, by BioLegend (1 mentions)
Facsaria cytometer, by BD (1 mentions)
2 protocols using type 4 collagenase
1
Isolation and Characterization of Immune Cells
2
Lung Cell Dissociation and Characterization
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The left lung lobe of each sample was dissociated in RPMI 1640 medium containing type IV collagenase (MP Biomedicals) and DNase I (Spectrum) using gentleMACS C tubes according to the manufacturer’s recommendations (Miltenyi Biotec). Cells were filtered through a 70-μm strainer, and red blood cells were lysed before blocking Fc receptors using anti-CD16/CD32 (BioLegend). Cell-specific proteins were labeled with CD11b-Alexa Fluor 488 (eBioscience), Ly6G-allophycocyanin (APC) (eBioscience), CD64-phycoerythrin (PE) (BioLegend), or SiglecF-peridinin chlorophyll protein (PerCP)-Cy5.5 (BD Biosciences) antibody or the appropriate isotype control antibodies. T cell subsets were stained with antibodies against CD3-PerCP-Cy5.5 (eBioscience), CD8-APC (eBioscience), and CD4-Alexa Fluor 488 (BioLegend). Stained cells were incubated in BD stabilizing fixative (BD Biosciences) and analyzed using a FACSAria cytometer (BD Biosciences). Results were analyzed using FlowJo software (TreeStar), and gating was performed based on fluorescence-minus-one controls. The gating strategy used to identify neutrophils, alveolar macrophages, and interstitial macrophages is shown in Fig. S7 in the supplemental material. T cells were gated as CD3+/CD4+ T helper cells and CD3+/CD8+ cytotoxic T cells.
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