5. Dissolve 30 μL of compound stock solution (2.5 mM in MQ
water) in 270 μL blood serum sample to a final concentration
of 0.25 mM (controls see Note 20).
6. Take one aliquot at timepoint zero and then after sequential
time points aliquots of 30 μL. Quench the reaction by adding
60 μL aqueous trichloroacetic acid (5% w/v) to the aliquot.
7. Vortex the quenched sample and subsequently, incubated for
15 min at 4
C.
8. Centrifuge the samples at 18,000 Â g at 4
C for 2 min.
9. Analyze supernatants by analytical RP-HPLC (UV 218 nm ) to
quantify compounds relative to timepoint zero, and also evaluated qualitatively by ESI-LC/MS to identify the compound
(m/z) in the sample.
3.4.3 In Vitro Toxicity
Measurement (MTT-Assay)
Previous treatments of stroke targeted the NMDA receptor and
thereby caused severe side effects. For an in vitro assessment of cell
toxicity of a compound we can perform an MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide)
assay. The MTT assay is a colorimetric assay that measures the
metabolic activity of a cell, by reducing of MTT in the mitochondria to a formazan product with purple color. The purple color can
be quantified by spectrometry, which correlates with cell viability.
The MTT assay was published by Sommer et al. with the compound
AVLX-147 instead of AVLX-144. AVLX-147 has an inverted and
stereoisomeric TAT solubility tag; nonetheless, the experimental
setup remains the same [25].
1. The experiment is performed on cortical neuronal cultures
after 7–8 days of in vitro in conditioned medium (500 μL per
well) at 37
C in a humidified atmosphere of 5% CO 2 .
2. For a positive control expose cultures to 10 μM MK-801
(Tocris Bioscience; Bristol, UK) for complete blockage of
NMDA receptors and complete neuroprotection.
3. For a negative control the cell medium was aspired and 525 μL
of 5 mM sodium azide in Hank’s Balanced Salt Solution
(HBSS) was added to the well, alternatively 100 mM acetic
acid can be used to achieve 100% cell death [26] (see Note 21).
4. Add in every well 25 μL of the compound as a 1 mM solution in
0.9% sterile saline.
5. For the NMDA concentration-response toxicity curve, NMDA
in concentrations from 1 μM to 1 mM was added to the wells.
6. Expose the cells to the compound by shaking at 37
C for 24 h.
7. Incubate the cells for 2 h with 0.5 mg mL
À1 MTT (see Note
22) at 37
C in a humidified atmosphere of 5% CO 2 .
PDZ Domain Peptide Inhibitors
173
water) in 270 μL blood serum sample to a final concentration
of 0.25 mM (controls see Note 20).
6. Take one aliquot at timepoint zero and then after sequential
time points aliquots of 30 μL. Quench the reaction by adding
60 μL aqueous trichloroacetic acid (5% w/v) to the aliquot.
7. Vortex the quenched sample and subsequently, incubated for
15 min at 4
C.
8. Centrifuge the samples at 18,000 Â g at 4
C for 2 min.
9. Analyze supernatants by analytical RP-HPLC (UV 218 nm ) to
quantify compounds relative to timepoint zero, and also evaluated qualitatively by ESI-LC/MS to identify the compound
(m/z) in the sample.
3.4.3 In Vitro Toxicity
Measurement (MTT-Assay)
Previous treatments of stroke targeted the NMDA receptor and
thereby caused severe side effects. For an in vitro assessment of cell
toxicity of a compound we can perform an MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide)
assay. The MTT assay is a colorimetric assay that measures the
metabolic activity of a cell, by reducing of MTT in the mitochondria to a formazan product with purple color. The purple color can
be quantified by spectrometry, which correlates with cell viability.
The MTT assay was published by Sommer et al. with the compound
AVLX-147 instead of AVLX-144. AVLX-147 has an inverted and
stereoisomeric TAT solubility tag; nonetheless, the experimental
setup remains the same [25].
1. The experiment is performed on cortical neuronal cultures
after 7–8 days of in vitro in conditioned medium (500 μL per
well) at 37
C in a humidified atmosphere of 5% CO 2 .
2. For a positive control expose cultures to 10 μM MK-801
(Tocris Bioscience; Bristol, UK) for complete blockage of
NMDA receptors and complete neuroprotection.
3. For a negative control the cell medium was aspired and 525 μL
of 5 mM sodium azide in Hank’s Balanced Salt Solution
(HBSS) was added to the well, alternatively 100 mM acetic
acid can be used to achieve 100% cell death [26] (see Note 21).
4. Add in every well 25 μL of the compound as a 1 mM solution in
0.9% sterile saline.
5. For the NMDA concentration-response toxicity curve, NMDA
in concentrations from 1 μM to 1 mM was added to the wells.
6. Expose the cells to the compound by shaking at 37
C for 24 h.
7. Incubate the cells for 2 h with 0.5 mg mL
À1 MTT (see Note
22) at 37
C in a humidified atmosphere of 5% CO 2 .
PDZ Domain Peptide Inhibitors
173