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LuminMax-C™ Easy to Use Attomole Luminometer |
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CELL QUANTIFICATION AND ATP TESTS
LuminMax-Q-Cell: Luminescent Cell Viability Assay 155 USD
1 vial LuminMax-ES, Luciferase and Substrate (luciferin) (lyophilized)
10 ml LuminMax-CB, Cell Buffer
100µl per assay (microwell),
the material is sufficient for 100 assays in 96-well plate
ATP: for Sensitivity and Standard Tests 175 USD
1 vial (400 µl, 100mM)
20µl for a series of dilution for a standard (calibration) test,
the material is sufficient for standard tests for 20 times
- The lyophilized LuminMax-ES and Cell Buffer should be stored at -20oC for
long-term (over one week) storage. For frequent use, it can be stored at 4oC;
however, it may lose some activity.
ATP Assay Protocol:
Purpose: To generate ATP standard curve
Material:
1. ATP 100mM
2. LuminMax-CB buffer
3. LumiMax-ES substrate (lyophilized)
4. Bottom clear 96 well microplate or strip: Must be compatible with the luminometer
5. Nuclease-free water
Reagent Preparation:
1. Thaw the LuminMax-CB Buffer and equilibrate to room temperature prior to use.
2. Equilibrate the lyophilized LuminMax-ES Substrate to room temperature prior to
use.
3. Transfer the appropriate volume of Buffer into the bottle containing Substrate to
reconstitute the lyophilized enzyme/substrate mixture. This is the Quantitative
Cell (or Signal) Reagent.
Experiment:
1. Prepare 1uM ATP in culture medium, buffer or nuclease-free water.
(100µl of 1µM ATP solution contains 10-10 moles ATP).
2. Prepare tenfold serial dilutions of ATP in culture medium, buffer or nuclease-free
water (e.g. 1µM to 10nM). (Dilute further for sensitivity test of the luminometer.)
3. Dispense 100µl of ATP solutions in microwell plate.
4. Add a volume of Q-Cell reagent equal to the volume of ATP.
5. Mix contents for 2 minutes on a shaker.
6. Incubate the plate at room temperature for 5 or 10 minutes.
7. Read luminescence with integration time of 100 ms or 1.0 second.
Example of ATP Standard Curve Assay
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Purpose: To Evaluate sensitivity with ATP assay using LuminMax-C |
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Material: |
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1. ATP: 100mM, 400ul, store at -20C, thawed, Store 4C. |
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Prepare (1)20ul + 1.98ml nuclease-free water = 1mM; |
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(2) 20ul of (1) +1.98 ml of water = 10uM |
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(3) 0.1ml of (2)+ 0.9ml water =1uM, 100ul 1uM ATP=10 -10moles ATP ; |
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(4) 20ul of (3)+1.98ml water =10nM; |
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(5) 20ul of (4)+1.98ml water = 100pM; |
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(6) 200ul of (5)+200ul water = 50pM; |
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(7) 200ul of (6) +200ul water = 25pM |
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2. |
Cell buffer, 10ml |
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Thaw and equilibrate the lyophilized substrate and buffer to RT. |
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Transfer all buffer into the substrate bottle. Mix. |
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Dispense 1.5ml per vial if needed. Store at 4C. |
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Experiment : |
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1. Dispense 100ul of ATP in the microwell. |
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2. Add 100ul of LuminMax-Q-Cell equal to the volume of ATP standard present in each well. |
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3. Mix for 2 minutes on orbital shaker. |
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4. Allow the plate to incubate at RT for 5 or 10 min. |
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5. Record luminescent on LuminMax for 100 ms or 1 second. |
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Result: |
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Microwell |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
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Concentration, 100ul |
0 |
25pM |
50pM |
100pM |
10nM |
100nM |
1uM |
10uM |
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5 min (100ms) |
3 |
9 |
20 |
57 |
141 |
590 |
5662 |
40497 |
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10 min (1 second)' |
40 |
137 |
243 |
433 |
1861 |
5925 |
57954 |
409921 |
Cell Assay Protocol:
Cell viability assay is similar to ATP assay protocol, instead of using ATP as analyte, cell sample is input to the microwell. Depend on the cell types; each cell may contain 10-15
- 10-18 mole of ATP. Once the cell titer (Cells per microwell) vs. Luminescence Signal is calibrated, the method can be used to correlate cell number with luminescent output.