Computational protocol: Single Dose Caffeine Protects the Neonatal Mouse Brain against Hypoxia Ischemia

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Protocol publication

[…] Randomization to caffeine or PBS treatment and sham or HI operation was performed at postnatal day 10 and samples were analyzed at 24h, 72h and 2 weeks after HI (total n = 52, treatment allocation is discernible in Fig 2).Damaged and undamaged brain hemispheres were gently homogenized using a loose fit glass homogenizer, and cell suspension passed through a 100um cell strainer prior to staining with fluorochrome conjugated antibodies. Splenocyte cell suspensions were prepared by gentle homogenization through a 100um cell strainer followed by red blood cell lysis with ACK buffer for 5 min at room temperature. For antibodies used see . Flow cytometric data was collected on a FACS Aria (BD Biosciences). 2.5x106 events were collected from each brain hemisphere.A data driven gating strategy was used with the FlowCore package for R [] as previously described in detail []. Debris was manually removed using a polygon gate in forward and side scatter. A curvfilter was used to filter out immune cell populations. Dead cells were excluded using DAPI stain. Negative population was selected as reference and cells with higher expression than that were regarded as positive cells. Median fluorescent intensities of the selected populations were used to discriminate between positive and negative populations. The same filter settings were used for each marker in all brain samples. After automated batching, all gate settings were visually confirmed and no manual adjustments were required. [...] Mann-Whitney U test was used to analyze data on morphology and behavior data. Statistical calculations were made in StatSoft, Inc. (2011) STATISTICA, version 10. Heatmaps were made in R version 2.15. […]

Pipeline specifications

Software tools flowCore, Statistica
Applications Miscellaneous, Flow cytometry
Organisms Mus musculus
Diseases Cerebrovascular Trauma, Hypoxia-Ischemia, Brain
Chemicals Caffeine, Phosphates