TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is part of the manuscript: <a href="http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.2000862" target="_blank">Gonçalves et al. Commensal bacteria and essential amino acids control food choice behavior and reproduction. Plos Biology. 2017 Apr 18.</a></p>
<p> </p>
<p>Holidic media (HM) were developed in collaboration with the laboratories of Linda Partridge at UCL and Matthew Piper at Monash University. </p>
<p> </p>
<p>The publications describing the holidic medium development are:</p>
<p> </p>
<p>Piper MDW, Blanc E, Leitão-Gonçalves R, Yang M, He X, Linford NJ, et al. A holidic medium for Drosophila melanogaster. Nat Methods. 2013;11: 100–105. doi:10.1038/nmeth.2731</p>
<p> </p>
<p>Piper MD, Soultoukis GA, Blanc E, Mesaros A, Herbert SL, Juridic P, et al. Matching Dietary Amino Acid Balance to the In Silico Translated Exome Optimizes Growth and Reproduction without Cost to Lifespan. Cell Metab. 2017;25: 610-621. doi:10.1016/j.cmet.2017.02.005</p>

[BEFORE_START]
<p>Make sure all solutions are available in enough quantities.</p>

[GUIDELINES]
<p> </p>
<p><img src="https://s3.amazonaws.com/pr-journal/iu3fw26.png" data-src="https://s3.amazonaws.com/pr-journal/iu3fw26.png" data-ofn="Screen Shot 2017-03-18 at 1.55.25 AM.png" /></p>
<p> </p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">In this publication, holidic media (HM) were prepared as described previously [1] using the HUNTaa formulation, with the exception of the HM used for pretreating axenic and gnotobiotic flies, for which we used an HM with an improved AA composition (FLYAA) [2]. In all experiments where we refer to all neAAs removal, L-Glutamate was still present in the diet in order to prevent any possible adverse effects in neuronal function.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong><em>Stock solutions for HUNTaa and FLYAA holidic media preparation</em></strong></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><em>1. Acetate buffer (10X) </em></p>
<ul style="text-align: justify;">
<li>to ~500 ml water, add 30 ml of acetic acid and 30 g KH<sub>2</sub>PO<sub>4</sub>, then slowly add 10 g NaHCO<sub>3</sub> as this will froth as CO<sub>2</sub> is released</li>
<li>fill up with milliQ water to just under 1000 ml </li>
<li>the final pH should be 4 otherwise use either HCl or NaOH to adjust to pH 4</li>
<li>fill up with milliQ water to exactly 1000ml</li>
<li>filter sterilize and store at 4ºC</li>
</ul>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><em>2. Cholesterol (66.67X) </em></p>
<ul style="text-align: justify;">
<li>weigh 1 g of cholesterol and fill up to 50 ml using absolute Ethanol</li>
<li>stir the mix for 15 min until everything is dissolved</li>
<li>aliquot into 15 ml Falcons and store at 4ºC</li>
<li>before use, warm to 40ºC for 30 min and vortex to re-dissolve the cholesterol</li>
</ul>
<p style="text-align: justify;"><em> </em></p>
<p style="text-align: justify;"><em>3. Trace solutions (CaCl<sub>2</sub>.6H<sub>2</sub>O, MgSO<sub>4</sub>, CuSO<sub>4</sub>.5H<sub>2</sub>O, FeSO<sub>4</sub>.7H<sub>2</sub>O, MnCl<sub>2</sub>.4H<sub>2</sub>O, Zn SO<sub>4</sub>.7H<sub>2</sub>O) (1000X)</em></p>
<ul style="text-align: justify;">
<li>add 1000 ml of water to 250 g CaCl<sub>2</sub>.6H<sub>2</sub>O</li>
<li>add 1000 ml of water to 250 g MgSO<sub>4</sub></li>
<li>add 1000 ml of water to 2.5 g CuSO<sub>4</sub>.5H<sub>2</sub>O</li>
<li>add 1000 ml of water to 25 g FeSO<sub>4</sub>.7H<sub>2</sub>O. To avoid problems with FeSO<sub>4</sub> rusting and dropping out of solution, aliquot and store at -20ºC. To use thaw aliquot once.</li>
<li>add 1000 ml of water to 1 g MnCl<sub>2</sub>.4H<sub>2</sub>O</li>
<li>add 1000 ml of water to 25 g Zn SO<sub>4</sub>.7H<sub>2</sub>O</li>
<li>filter sterilize each solution except for FeSO<sub>4, </sub>aliquot into 2 ml Eppendorf tubes and store at -20ºC</li>
<li>thaw every aliquot only once, the solution keeps for up to a week when stored at 4ºC</li>
</ul>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><em>4. Nucleic acids and lipid-related metabolites (125X)</em></p>
<ul style="text-align: justify;">
<li>weigh 6.25 g choline chloride, 0.63 g myo-inositol, 8.13 g inosine and 7.5 g uridine and fill up to 1000 ml using milliQ water</li>
<li>filter sterilize, aliquot into 50 ml Falcons and store at 4ºC wrapped in aluminium foil to minimize light exposure</li>
</ul>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><em>5. Vitamins (71.5X) </em></p>
<ul style="text-align: justify;">
<li>weigh 0.100 g thiamine (aneurin), 0.050 g riboflavin, 0.600 g nicotinic acid, 0.775 g Ca pantothenate, 0.125 g pyridoxine and 0.010 g biotin and fill up to 1000 ml using milliQ water</li>
<li>filter sterilize, aliquot into 15 ml Falcons and store at -20ºC</li>
<li>thaw every aliquot only once, the solution keeps for up to a week when stored at 4ºC wrapped in aluminium foil to minimize light exposure</li>
</ul>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><em>6. Folic acid (1000X)</em></p>
<ul style="text-align: justify;">
<li>to dissolve 0.5 g of folic acid in 1000 ml of milliQ water add NaOH (2M) dropwise</li>
<li>filter sterilize, aliquot into 2 ml Eppendorf tubes and store at -20ºC</li>
<li>thaw every aliquot only once, the solution keeps for up to a week when stored at 4ºC wrapped in aluminium foil to minimize light exposure</li>
</ul>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><em>7. Essential amino acids HUNTaa diet (10X)</em></p>
<ul style="text-align: justify;">
<li>dissolve 5.2 g phenylalanine, 4 g histidine, 7.6 g lysine, 3.2 g methionine, 3.2 g arginine, 8 g threonine, 11.2 g valine and 2 g tryptophan into 350 ml milliQ filtered water and vigorously stir until it is dissolved. The solution is close to saturation and therefore requires at least 2 hours to dissolve and may need to be warmed up to 65-75ºC while stirring vigorously. Isoleucine and leucine are handled separately as their solubility is very low.</li>
<li>wash the sides of the flask with a plastic pipette to ensure everything goes into solution.</li>
<li>
<p>adjust pH to 4.5 using 5 M HCl and fill up to 400 ml with milliQ filtered water</p>
</li>
<li>
<p>filter sterilize and store at 4ºC wrapped in aluminium foil to minimize light exposure, the solution keeps for up to two months.</p>
</li>
</ul>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><em>8. Non essential amino acids HUNTaa diet (10X)</em></p>
<ul style="text-align: justify;">
<li>dissolve 14 g alanine, 0.2 g cysteine, 6.8 g aspartic acid, 12.8 g glycine, 6.8 g asparagine, 6 g proline, 10 g glutamine and 7.6 g serine into 350 ml milliQ filtered water and vigorously stir until it is dissolved. The solution is close to saturation and therefore requires at least 2 hours to dissolve and may need to be warmed up to 65-75ºC while stirring vigorously. Tyrosine is handled separately as its solubility is very low. Glutamic acid is added separately for flexibility - if an amino acid or a group of amino acids are omitted, the amount of glutamic acid can be adjusted to compensate for the loss of nitrogen. </li>
<li>wash the sides of the flask with a plastic pipette to ensure everything goes into solution.</li>
<li>adjust pH to 4.5 using 1 M HCl and fill up to 400 ml with milliQ filtered water</li>
<li>filter sterilize and store at 4ºC wrapped in aluminium foil to minimize light exposure, the solution keeps for up to two months.</li>
</ul>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><em>9. Essential amino acids FLYAA diet (10X)</em></p>
<ul style="text-align: justify;">
<li>
<p>dissolve 6.66 g phenylalanine, 4.32 g histidine, 9.02 g lysine, 3.98 g methionine, 10.78 g arginine, 7.32 g threonine, 7.94 g of valine and 2.12 g of tryptophan into 350 ml milliQ filtered water and vigorously stir until it is dissolved. The solution is close to saturation and therefore requires at least 2 hours to dissolve and may need to be warmed up to 65-75ºC while stirring vigorously. Isoleucine and leucine are handled separately as their solubility is very low.</p>
</li>
<li>wash the sides of the flask with a plastic pipette to ensure everything goes into solution.</li>
<li>
<p>adjust pH to 4.5 using 5 M HCl and fill up to 400 ml with milliQ filtered water</p>
</li>
<li>
<p>filter sterilize and store at 4ºC wrapped in aluminium foil to minimize light exposure, the solution keeps for up to two months.</p>
</li>
</ul>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><em>10. Non essential amino acids FLYAA diet (10X)</em></p>
<ul style="text-align: justify;">
<li>dissolve 7.28 g alanine, 7.74 g aspartic acid, 5.08 g glycine, 6.8 g asparagine, 6.46 g proline, 7.42 g glutamine and 9.12 g serine into 350 ml milliQ filtered water and vigorously stir until it is dissolved. The solution is close to saturation and therefore requires at least 2 hours to dissolve and may need to be warmed up to 65-75ºC while stirring vigorously. Tyrosine is handled separately as its solubility is very low. Glutamic acid is added separately for flexibility - if an amino acid or a group of amino acids are omitted, the amount of glutamic acid can be adjusted to compensate for the loss of nitrogen.</li>
<li>wash the sides of the flask with a plastic pipette to ensure everything goes into solution.</li>
<li>adjust pH to 4.5 using 1 M HCl and fill up to 400 ml with milliQ filtered water</li>
<li>filter sterilize and store at 4ºC wrapped in aluminium foil to minimize light exposure, the solution keeps for up to two months.</li>
</ul>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><em>11. Glutamic acid for HUNTaa and FLYAA diets (100 mg/ml)</em></p>
<ul style="text-align: justify;">
<li>dissolve 20 g of glutamic acid in 200 ml of milliQ filtered water and stir until it is dissolved. If using the free acid, glutamic acid will only go into solution when NaOH is added dropwise.</li>
<li>filter sterilize and store at 4ºC wrapped in aluminium foil to minimize light exposure, the solution keeps for up to two months.</li>
</ul>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><em>12. Cysteine for FLYAA diet (50 mg/ml)</em></p>
<ul style="text-align: justify;">
<li>dissolve 10 g of cysteine in 200 ml of milliQ filtered water and stir until it is dissolved.</li>
<li>filter sterilize and store at 4ºC wrapped in aluminium foil to minimize light exposure, the solution keeps for up to two months. </li>
</ul>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><em>13. Nipagin (100g/l)<br /></em></p>
<ul style="text-align: justify;">
<li>dissolve 10 g of nipagin in 100 ml of milliQ filtered water and stir until it is dissolved.</li>
<li>store at room temperature in aluminium foil to minimize light exposure. </li>
</ul>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>References</strong></p>
<p style="text-align: justify;"> </p>
<p>1. Piper MDW, Blanc E, Leitão-Gonçalves R, Yang M, He X, Linford NJ, et al. A holidic medium for Drosophila melanogaster. Nat Methods. 2013;11: 100–105. doi:10.1038/nmeth.2731</p>
<p> </p>
<p>2. Piper MD, Soultoukis GA, Blanc E, Mesaros A, Herbert SL, Juridic P, et al. Matching Dietary Amino Acid Balance to the In Silico Translated Exome Optimizes Growth and Reproduction without Cost to Lifespan. Cell Metab. 2017;25: 610-621. doi:10.1016/j.cmet.2017.02.005</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.