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How to make recording medium

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    Shin Yamazaki
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    I have been asked many questions regarding with bioluminescence recording. Two most commonly asked questions are how to make recording medium and selection of incubator. I decided to post those answers.

    How to make recording medium
    [Recording medium]
    1. DMEM powder Cellgro (90-013) 12.77g
    2. L-Glutamine (200 mM/water; Sigma G7513) 20 ml
    3. NaHCO3 (7.5%/water; Sigma G8761) 4.7 ml
    4. HEPES (1M/water; Sigma H0887) 10 ml
    5. Penicillin-Streptomycin (100X/0.9% saline; Sigma P0781) 2.5ml

    Put #1 into Milli-Q water and mix well
    Add #2-4 and measure up with 1L volumetric flask
    Add #5
    Filter with Millipore Stericup Sterile Vacuum Filter System.

    It is very important to know if the solution is made of water or saline. If the solution is made of water, add the solution BEFORE you measure up with 1L volumetric flask. If the solution is made of saline add the solution AFTER you measure up with volumetric flask. I don?t autoclave Milli-Q water, because of a previous bad experience (most likely toxic material contaminated by shared autoclave). I don?t adjust the pH. pH should be stable in a few days. If you have osmometer, it is better to check osmolality.

    NOTE: If you are using a bright sample, premade 1X liquid medium with phenol red without NaHCO3 can be used. I know several researchers are using medium with phenol red. If you need to use low glucose medium, please see the book chapter I wrote with Joe Takahashi, https://www.ncbi.nlm.nih.gov/pubmed/15817295)

    [Luciferin Stok]
    Beetle Luciferin Potassium Salt (Promega E160B, 50 mg).
    Add 1.7 ml cell culture grade water.
    Aliquot 50 ul in each tube.

    [Final Recording Medium]
    1. 50 ml Recording medium
    2. FBS or B27
    3. Luciferin

    • This topic was modified 3 years, 3 months ago by Shin Yamazaki.
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