Alamar Blue Assay
Background Info:
alamarBlue is a bioassay used to quantify cell viability. The nontoxic dye both fluoresces and changes color when it undergoes REDOX reactions as a result of cellular respiration and proliferation. (Dye not incorporated into the cells will also change color due to the media being reduced as well.)
Oxidized form = non-fluorescent and blue Reduced form = fluorescent and red/pink
The results are measured as absorbance at 570nm and 600nm on the spectrophotometric plate reader. Graphable numbers are calculated using the excel template, where 600nm is used as a reference measurement.
Things to know before starting:
a. alamarBlue is light sensitive, so it must be stored in the dark (can be frozen indefinitely) and lights should be turned off when working with it.
b. If your cell treatment involves GLT (glucolipotoxic media), it will interfere with the absorbance readings and a slightly different protocol should be used, which will be detailed below.
c. Since the dye is non-toxic, cells can be placed in new media and returned to culture if so desired.
d. The dye has a weird viscosity! Make sure to double check your multichannel pipettes are filled equally while working with it.
Stock Recipe:
- 3mg Resuzurin sodium salt (dry goods shelf)
- 27.15ml 1x PBS
- Mix, filter, aliquot and freeze
**Pre-made aliquots are currently stored in the dartboard freezer and can be used at a concentration of 1 uL alamar: 10 uL media. (04/2025)
Step 1: Plate cells
Plate cells in 96-well plates. As you do so, make sure to leave room for the following on each plate:
1. One column left COMPLETELY EMPTY (no cells or media). This will be used to measure background absorbance during data quantification.
2. One column with NO CELLS. If you are not treating your cells with GLT (glucolipotoxic) media, this column can be filled with plain media during initial treatment and incubated with the rest of the cells. If you are treating your cells with GLT media, leave this column empty for now.
Protocol for GLT treatments:
Amounts given are for one 96-well plate. (Amounts for three 96-well plates are given in parentheses).
1. Turn off lights in the hood and hood room (I usually just turn off one set so I can actually see what I’m doing—just make sure to minimize the amount of time it’s exposed to direct light)
2. Thaw aliquot of alamarBlue and warm media in water bath
3. Vortex thawed alamarBlue to mix well
4. Mix 1ml (3 mL) stock alamarBlue to 10ml (30 mL) of warm media (I find this is often a bit too much media for my treatments—this is a general number, so feel free to play around with it).
5. Aspirate media from 1 column at a time and add 100ul of alamarBlue media into each well using multichannel pipetter (can also aspirate whole plate then add the alamar blue to each column if you’re fast enough—I find this to be much less annoying).
6. Add alamar blue media into cell-free column. Make sure to still leave at least one column completely empty.
7. Put plate(s) in incubator, protected from light. Results can be read anywhere from 1 to 4 hours. For the GLT specifically, I recommend reading it at the 1 to 2 hour mark as the cells will start to recover and skew your results.
8. To read the results, take plates to the plate reader of your choice (Berges lab currently). Read plates with the lids off. Use the Tessem lab alamar blue absorbance protocol (absorbance is read at 570 and 600 nm). You can also measure florescence (at excitation 560 nm, emission 590 nm), which is supposed to be more accurate, but I haven’t figured out how to interpret the data from that yet and I don’t particularly want to.
9. Freeze plates or return them to normal culture media if you want to do more stuff with the cells, because you hypothetically can.
Protocol for non-GLT treatments:
Amounts given are for one 96-well plate. (Amounts for three 96-well plates are given in parentheses).
1. Turn off lights in the hood and hood room (I usually just turn off one set so I can actually see what I’m doing—just make sure to minimize the amount of time it’s exposed to direct light)
2. Thaw aliquot of alamarBlue and warm media in water bath
3. Mix 1,350 uL (4,050 uL) 1x PBS with 150 uL (450 uL) of the stock alamar blue dye. I like to use a cover over the dye container after mixing it to protect it from light more. (Amounts given overshoot the estimated mark by a factor of 1.5 due to the small amounts you’re working with and the slipperiness of the solution).
4. Do not aspirate the media from your plates. Instead, add 10 uL of the PBS/alamar solution to each well using the multichannel pipettor. Make sure to also add dye to the media-only column, and leave a column completely empty. (Tip: use the electronic multichannel pipettor or else your life will be a living hell. The mechanical ones don’t work well with the dye and will bring up unequal amounts plus your arm will hurt. Trust me.)
5. Tap/swirl plate to mix so that dye is mixed evenly with media. You can also pipette to mix with a 100 uL multichannel (make sure not to cross contaminate treatment groups. You will also need 5 billion pipette tips so be prepared for that). If you pipette to mix, don’t eject all the air from it (it will make nasty nasty bad bubbles) and use an electronic pipettor if you value a pain-free hand and forearm.
6. Put plate(s) in incubator, protected from light. Results can be read anywhere from 1 to 4 hours. Between 2-3 hours is usually a solid sweet spot.
7. To read the results, take plates to the plate reader of your choice (Berges lab currently). Read plates with the lids off. Use the Tessem lab alamar blue absorbance protocol (absorbance is read at 570 and 600 nm).
8. Freeze plates or return them to normal culture media if you'd like to continue to use the cells.