Summary of advantages and disadvantages of using BDNF ELISA, BDNF-eGFP, BDNF-pHluorin.
|BDNF ELISA||Quantify levels of BDNF in homogenized tissue, or levels of released BDNF if using ELISA in situ ||Genetic manipulations are unnecessary. Endogenous BDNF levels can be quantitatively measured with pg sensitivity. proBDNF-specific ELISA kits are available. If using ELISA in situ, the amount of released BDNF can be accurately quantified, and tissue need not be destroyed.||Cannot identify sites of BDNF release, and cannot observe trafficking of BDNF vesicles. ELISA is performed over the course of hours, and changes in BDNF cannot be observed in real time. ELISA in situ inhibits BDNF-TrkB signaling.||[13, 19, 20, 22, 24, 51, 57, 59, 65]|
|BDNF-eGFP||Visualize BDNF trafficking and release in real time||BDNF vesicle dynamics can be observed throughout the cell. Sustained vesicle release can be observed by a relative decline in fluorescence. Can be used in conjunction with western blot to determine relative levels of mBDNF and proBDNF. Downstream signaling of BDNF-eGFP release is similar to that of BDNF.||cDNA plasmids for BDNF-eGFP must be introduced by transfection or viral transduction, which can be harsh on cells, and/or can lead to artificial overexpression of BDNF. Cannot quantify absolute levels of released BDNF.||[11, 12, 28–31, 33, 35, 36]|
|BDNF-pHluorin||Visualize BDNF vesicle release kinetics in real time||The pH sensitivity of pHluorin enables discrimination between sustained and transient vesicular fusion, which is indicative of how much BDNF is diffusing out of each vesicle. Downstream signaling of BDNF-pHluorin release is similar to that of BDNF.||Because fluorescence is quenched at low pH, BDNF-pHluorin is difficult to track while inside of acidified vesicles, making this tool unsuitable for BDNF trafficking studies. The same disadvantages as BDNF-eGFP also apply.||[10, 85]|