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[BEFORE_START]
<p><span style="font-weight: 400;">Pre-made Alamar Blue (Resazurin) solutions from companies (e.g. </span><a href="https://www.thermofisher.com/order/catalog/product/DAL1025" target="_blank" rel="noopener noreferrer"><span style="font-weight: 400;">alamarBlue™ Cell Viability Reagent</span></a><span style="font-weight: 400;">)  are relatively expensive for long-term and common use. Therefore, ordering it in powder format is the cheapest and the most feasible way to go for extended use cases. 1 g powder (e.g. </span><a href="https://www.sigmaaldrich.com/catalog/product/sigma/r7017?lang=en&amp;region=US" target="_blank" rel="noopener noreferrer"><span style="font-weight: 400;">Resazurin sodium salt #R7017</span></a><span style="font-weight: 400;">) should last for a year or two even if it is frequently used.</span></p>
<p> </p>
<p><span style="font-weight: 400;">The final working concentration for Resazurin is 44 uM and our goal is to prepare a 10X solution for use in cell culture (e.g. for 200 ul of media/cells, we will add 20 ul of the dye). Directly preparing this is not feasible (due to the amount of PBS required) so we will first prepare a 1000X stock solution which we will further dilute as needed.</span></p>
<p> </p>
<p><span style="font-weight: 400;">For 1000X Stock (44 mM):</span></p>
<ul>
<li style="font-weight: 400;"><span style="font-weight: 400;">Weigh 5 gram of Resazurin powder</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Resolve the powder in 500 ml PBS (this will require vigorous shaking/mixing for a few times so make sure you don’t have any residues in the solution)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Sterile filter the solution and prepare 10 aliquots (each 50 ml)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Store the 1000X stocks at -20</span><span style="font-weight: 400;">°C </span><span style="font-weight: 400;">(either wrap each one with aluminum foil or use a cardboard box to limit light exposure for long term)</span></li>
</ul>
<p> </p>
<p><span style="font-weight: 400;">For 10X Stock (440 uM):</span></p>
<ul>
<li style="font-weight: 400;"><span style="font-weight: 400;">Add 500 ul of 1000X stock onto 50 ml of PBS in a falcon tube (usually makes sense to prepare 4-5 of these)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Wrap the falcon tube with aluminum foil so that it is minimally exposed to sunlight</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Store 10X working solutions at 4</span><span style="font-weight: 400;">°C</span><span style="font-weight: 400;"> (they should be good for up to 6 months)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Can also store one 1000X stock at 4</span><span style="font-weight: 400;">°C</span><span style="font-weight: 400;"> for convenience (if planning to assay frequently)</span></li>
</ul>

[GUIDELINES]
<ul>
<li style="font-weight: 400;"><span style="font-weight: 400;">Always make sure you have at least 4 replicates</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Always make sure to have blank media control (ideally on the same plate but having another plate with only media in it is OK, too)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Plate readers are relatively less accurate for the most outer wells so do not use them specifically for a condition alone (it might bias your results). Having a replicate for each of your conditions in either of the most outer wells is OK.</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">It is much easier to work in batches via multichannel pipettors and make use of the reagent cuvette</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">The dye is sensitive to light/temperature. No need to go crazy about this but make sure you don’t leave the plate out for more than 10 minutes before measuring</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Do not go over 200 ul in total when preparing your cultures</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Plan carefully and remember to add the dye a day (24 hours) before your endpoint assay</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">The assay is not that accurate &gt; 300,000 cells per well or &lt; 1,000 cells per well so make sure you seed the cells in such a way that you will hit the reliable cell counts on the day of your measurement</span></li>
</ul>

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