Fig 1: Quality control for collection and handling of murine CSF samples.A, quality control scheme for CSF collection and handling. If a leakage of CSF occurs on the dura, the sample has to be discarded. After the collection, samples are centrifuged at 2000g for 10 min at 4 °C. If a red pellet is visible, discard the sample. If there is no pellet, aliquot the CSF in low protein binding tubes (i.e. 0.5 ml protein lobind tubes, Eppendorf). Add 10 μl of PBS to the emptied collection tube, which might contain pelleted cells, and check it in a Neubauer chamber. Keep the CSF sample if you have less than 40 cells per μl. Alternatively, i.e. if samples were already collected and stored without performing cell counting, 1 μl of sample can be used for a hemoglobin ELISA (#ab157715, Abcam). Keep the sample if values are below the LOD. B, microscopical control and simulation of blood-contamination at Neubauer chamber. The left figure shows a reconstituted CSF pellet under a 10x and 20x (magnifications to the right) objective. This pellet contains the particles or cells that were in the collected CSF, before centrifugation. Note that apart from few debris (arrows and arrowheads), which is presumably due to dura puncture and is removed by centrifugation, the CSF is largely devoid of cells. Next to it, the simulation of blood contamination with 1% and 0.01% are shown in a Neubauer chamber (under a 10x objective and 20x insert magnification, arrowheads point at erythrocytes). The cell counting results of CSF-cells (in its reconstituted pellet of WT (n = 5) and 5xFAD (n = 5) animals) of 1% blood, 0.01% blood (n = 5, mean ± 95%CI) and the murine CSF (n = 9, mean ± 95%CI) show a 200-fold lower cell content than the 0.01% blood contamination cut-off criteria. C, ELISA-based quantification of hemoglobin (Hb) in CSF of WT (n = 5) and 5xFAD (n = 5) animals shows levels below the detection limit of the method for 9/10 samples; for comparison, our simulation experiments of blood contamination show detectable Hb for 1% contamination, and below detection limit for 0.01% (minimal accepted level of CSF contamination with blood (22)) Normal mouse plasma contained 38.5 ± 14.3 mg/L of free Hb.