3 Methods
This present protocol has been optimized for studying metabolic
fluxes in primary neuronal cultures, using a Seahorse XFe24. How-
ever, this assay can be run on different cell types. In this case all the
procedures can be easily adapted and optimized from the present
protocol.
Carry out all steps at room temperature (RT) unless otherwise
specified.3.1 Cell Culture All the procedures for cell plating must be carried under a laminar
flux to guarantee sterile conditions. For primary neuronal culture,
plate coating is done the day before cell plating (seeNote 5). Here
we will not detail the procedures for obtaining primary neuronal
culture, as it is beyond the scope of this protocol, but the reader can
find further details in Domise et al. [17].
Plate Coating
- Unpack the box containing the XFe24 multi-well cell plate
provided in the kit (Fig.1a)(seeNote 6). - Aliquot in each well 70 μlof15μg/ml poly L-ornithine
solution. - Incubate the plate for 45 min at RT.
- Substitute the polyL-ornithine with 70μlof4μg/ml polyL-
laminin solution. - Incubate the plates overnight (ON) at 37C.
Fig. 2Optimization of FCCP concentration to induce maximal respiration. Different concentrations of FCCP
(0.05, 0.1, 0.175, 0.5μM) were used to induce maximal respiration in primary neurons. (a) OCR (pmoles/min)
is monitored at baseline (basal respiration), after the injection of oligomycin and in response to the different
concentrations of FCCP. (b) Spare respiratory capacity expressed as the difference between maximal OCR
upon FCCP injection and basal respiration. Cells were tested between 14 and 17 days in vitro (DIV). Data are
meanSD ofn¼3–4. One-way ANOVA, post hoc test Bonferroni **p<0.05, ***p<0.01
294 Claudia Marinangeli et al.