Authors: Alexi Nott , James D. Robinson & Antonella Riccio
In situ imaging of nitric oxide (NO) in cells can be performed using 4-amino-5-methylamino-2’,7’-difluororescein diacetate (DAF-FM) (1). The ability to visualise NO production upon activation of endogenous NOS isoforms in a culture system is becoming increasingly important for studying NO signalling.
We have detected NO accumulation in embryonic cortical neurons upon activation of endogenous nNOS following stimulation with the neurotrophin brain-derived neurotrophin factor (BDNF) (2). Importantly, we were able to detect NO accumulation within both the cytoplasm and the nucleus. This was achieved through culturing cortical neurons in low serum and the use of a DAF-FM-DA probe in combination with a CellTracker Red CMTPX probe.
Unless specified, reagents were obtained from Sigma, UK.
- Poly-D-Lysine (Sigma #P1024)
- Laminin (BD Bioscience #354232)
- Papain (Worthington #PAP LS003126)
- Cysteine-HCL (Sigma #C7477)
- Digestion Solution: 81.75mM Na2SO4, 30mM K2SO4, 5.8mM MgCl2, 250nM CaCl2, 1mM Hepes pH 7.4, 20mM glucose, 0.001% phenol red. Sterile Filter.
- Dissection Media: Hanks Balanced Salts (HBSS; Sigma #H6136), 2.5mM Hepes pH 7.4, 35mM glucose, 1mM CaCl2, 1mM MgSO4, 4mM NaHCO3. Sterile Filter.
- Cortex Media: MEM media (Gibco #31095), 10% Foetal Bovine Serum (FBS; Invitrogen #16000044), 5% Horse serum (HS; Hyclone #SH30074.03), 1mM Glutamine (PAA #M11-004).
- Tripan Blue Solution (Sigma #T8154)
- DL-2-amino-5-phosphonovaleric acid (DL-AP5; Tocris #105)
- 4-amino-5-methylamino-2’,7’-difluororescein diacetate (DAF-FM DA; Molecular Probes #D23842)
- CellTracker Red CMTPX (Invitrogen #C34552)
- Four-well plates (Nunc #176740)
- 13mm Glass Coverslips, Thickness No. 1.5 (VWR #631-0150)
- 40 µM Cell Strainer (BD Falcon #352340)
- Hemocytometer chamber
- 1. Treat coverslips with 0.1mg/ml poly-D-lysine and 0.01mg/ml laminin and leave at 37oC overnight. The next day remove the coating solution and dry thoroughly.
- 2. Prepare the digestion, dissection and cortex media’s as described in Reagents. Place the dissection media on ice, and the cortex media at 37oC.
- 3. Prepare the papain solution on the day of culture: Add 1ml 20mM cysteine-HCl and 400U papain to 9ml digestion solution. Warm the solution at 37oC for 20 minutes and then neutralize to pH 7.0 using 0.1M NaOH. Filter sterilize and keep at room temperature.
Isolation of rat embryonic cortices
- 4. Sacrifice E17 rat according to institutional ethically approved procedures.
- 5. Immediately remove the uterus and place in dissection solution on ice.
- 6. Isolate individual embryos, decapitate and remove the whole brain, and place in fresh dissection media.
- 7. Dissect the cortices using a dissection microscope being careful to remove the meninges. Collect the cortices in a 50ml tube containing ice-cold dissection solution.
- 8. Remove dissection solution from the cortices and add 10mls papain solution.
- 9. Leave at 37oC for 25 minutes, gently swirling the cortices every 5 minutes. Do not shake the tubes during the last 5 minutes of digestion.
- 10. Remove the papain solution carefully as cortices become ‘sticky’. Immediately add 1ml FBS to inhibit any remaining papain.
- 11. Wash the cortices by gentle addition and removal of 10ml warm cortical media. Repeat washes four times.
- 12. Dissociate the cortices in 10mls cortical media by passing through a 10ml plastic pipette 8 times.
- 13. Pass the dissociated culture through a 40µM cell strainer.
- 14. Estimate the cell number using a hemocytometer chamber and dilute the culture to 0.5M cells per ml.
- 15. Plate 200,000 cells per coated coverslip in a 4-well plate and place in a cell incubator at 37oC and 10% CO2.
- 16. Perform DAF imaging on cortical neurons at 4 days in vitro. Starve cells 16 hours prior to the following protocol in media containing 3% serum and 50µM DL-AP5.
- 17. Load cells with the probe by replacing media with DMEM containing 1mM glutamine, 50µM DL-AP5 and 10 µM DAF-FM-DA and left at 37oC for 30 minutes. Use DMEM free of phenol red as it may affect the fluorescence.
- 18. Wash the cells three times using media to remove excess probe. Replace the cells at 37oC for 15 minutes. This allows intracellular de-esterification of the probe to form the cell impermeable DAF-FM.
- 19. Treat the cells as desired. NO production usually occurs rapidly, within minutes. However, if longer time courses are required then return the cells to 37oC.
- 20. Upon completion of treatment, replace the media with DMEM containing 2.67ng/ml CellTracker Red CMTPX for 30 minutes to allow visualization of cell morphology.
- 21. Wash the cells once in PBS containing Hoescht for 5 minutes.
- 22. Wash twice with PBS alone and fix in 4% PFA at room temperature for 15 minutes.
- 23. Immediately mount the coverslips and image within 24 hours using a confocal microscope at an excitation/emission maxima of 495/515nm.
We have demonstrated that stimulation of rat embryonic cortical neurons with the neurotrophin BNDF, induces production of NO (ref. 2). Neuronal production of NO occurs within minutes of BDNF stimulation and is found localised within the nucleus, as well as the cytoplasm. The nNOS-specific inhibitor, NO-propyl-L-arginine, inhibits BDNF stimulated production of NO. Therefore, neurotrophin-dependent NO production requires the Ca2+-dependent activation of endogenous nNOS.
The protocol presented here allows the visualisation of nitric oxide accumulation in the nucleus. The use of CellTracker CMTPX allows the visualisation of neuronal morphology without employing laborious immunostaining technique. This is of particular importance due to the instability of the fluorescent benotrialzole produced upon reaction of NO with DAF-FM. CellTracker Red CMTPX has the additional advantage of being nuclear impermeable. This is critical to allow confident imaging of NO accumulation within the nucleus.
The protocol permits detection of neurotrophin-dependent activation of endogenous nNOS. E17 cortical neurons kept in media containing serum for 4 days in vitro will already express glutamatergic receptors. Experimental procedures, such as loading the cells with probe followed by repeated washes may lead to stimulation of neurons and subsequent excessive Ca2+ signalling and nNOS activation. Therefore, starving cortical cultures in media containing DL-APV for 16 hours is critical to reduce background levels of NO production.
- Kojima, H., Nakatsubo, N., Kikuchi, K., Kawahara, S., Kirino, Y., Nagoshi, H., Hirata, Y., Nagano, T. Detection and imaging of nitric oxide with novel fluorescent indicators: diaminofluoresceins. Anal. Chem. 70, 2446-2453 (1998).
- Nott, A., Watson, P.M., Robinson, J.D., Crepaldi, L., Riccio, A. S-nitrosylation of histone deacetylase 2 induces chromatin remodelling in neurons. Nature 455, 411-415 (2008).
Figure 1.: Imaging nuclear accumulation of nitric oxide in embryonic cortical neurons.
Cortical neurons were loaded with 10µM DAF-FM for 30 minutes prior to treatment with BDNF (100ng/ml for 10 minutes). Cells were stained with CellTracker red and Hoescht to allow visualisation of cell morphology and nuclei respectively. Nitric oxide is detected upon reaction with DAF-FM at an emission/excitation maxima of 495/515nm. Images were captured using a Leica DM2500 confocal microscope. Scale bar is 25µM.
S-nitrosylation of histone deacetylase 2 induces chromatin remodelling in neurons, Alexi Nott, P. Marc Watson, James D. Robinson, Luca Crepaldi, and Antonella Riccio, Nature 455 (7211) 411 - 415 18/09/2008 doi:10.1038/nature07238
Alexi Nott , James D. Robinson & Antonella Riccio, MRC Laboratory for Molecular and Cell Biology, and Department of Neuroscience, Physiology and Pharmacology, University College London, London WC1E 6BT, UK
Source: Protocol Exchange (2008) doi:10.1038/nprot.2008.235. Originally published online 4 November 2008.