Computational protocol: Glucocorticoids limit acute lung inflammation in concert with inflammatory stimuli by induction of SphK1

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Protocol publication

[…] Blood samples were obtained by cardiac puncture. A 100-μl aliquot of blood was collected into 900 μl Alsever's solution and then centrifuged at 350g for 10 min at 4 °C. The supernatant was removed, the cells were treated twice with erythrocyte lysis buffer, washed with 0.1% BSA in PBS and then resuspended in the reflux. Cell counts were determined with the aid of a Neubauer haemocytometer.For flow cytometric analysis, Fc receptor blockade was performed by incubation with TruStain fcX (anti-mouse CD16/32; clone: 93; cat. no. 101320; 1:2,000). Subsequently, the cells were stained with the following monoclonal antibodies: PerCP/Cy5.5 anti-mouse CD3 (clone: 17A2; cat. no. 100218; 1:500), APC anti-mouse/human CD45R/B220 (clone: RA3-6B2; cat. no. 103212; 1:800), PE anti-mouse CD49b (clone: DX5; cat. no. 108908; 1:400), FITC anti-mouse F4/80 (clone: BM8; cat. no. 123108; 1:500), APC/Cy7 anti-mouse Ly-6G/Ly-6C (Gr-1; clone: RB6-8C5; cat. no. 108424; 1:2000), PE/Cy7 anti-mouse CD11c (cat. no. 117318; 1:500), PE anti-mouse CD115 (cat. no. 135505; 1:500). All the antibodies were obtained from BioLegend (Uithoorn, The Netherlands). Data were acquired on a BD FACS Canto II flow cytometer (BD Biosciences) and were analysed using FlowJo software (Tree Star). [...] Results are presented as mean±s.e.m. Statistical analyses were performed with one-way analysis of variance (ANOVA) for comparison, followed by Tukey's post hoc within one group. For the comparison of two groups (genotypes), one-way analysis of variance followed by Tukey's post hoc test for multiple comparisons was performed. Statistical analyses were performed using the GraphPad Prism software (GraphPad Software) and sample size was chosen with G*Power3.1 (University Düsseldorf). Outlying sample exclusion criteria were done with GraphPad Prism Outlier Calculator. Data were considered statistically significant: *P<0.05, **P<0.01 and ***P<0.001. […]

Pipeline specifications

Software tools FlowJo, G*Power
Applications Miscellaneous, Flow cytometry
Diseases Pneumonia, Acute Lung Injury
Chemicals Sphingosine