Calcium ion in the sample bind to Methyl Thymol Blue (MTB) in alkaline solution and form blue complex. Calcium content can be calculated by measuring the OD value at 610 nm.

1.  Avoid calcium contamination in the experiment. Disposable 96-wells plate is recommended.

2.  Severe hemolysis, jaundice and lipidemia will affect the experiment result.

3.  It is recommended to use heparin anticoagulant to prepare plasma sample instead of calcium chelator and oxalate anticoagulant. 

4.  Use deionized water as homogenized medium to avoid calcium contamination when preparing tissue/cell homogenates.

               [Note]: A-H, standard wells; S1-S80, sample wells.

1.    For serum(plasma) and other liquid sample:

1)    Standard tube: Take 10 μL of Standard solution with different concentrations to the wells.

Sample tube: Take 10 μL of sample to the wells.

2)    Add 250 μL of working solution 1 into the wells of Step 1.

3)    Mix fully and stand for 5 min at room temperature.

4)    Measure the OD value at 610 nm with Microplate Reader

2.    For tissue/cell homogenate sample:

1)    Standard tube: Take 10 μL of Standard solution with different concentrations to the wells.

Sample tube: Take 10 μL of sample to the wells.

2)    Add 250 μL of working solution 2 into the wells of Step 1.

3)    Mix fully and stand for 5 min at room temperature.

4)    Measure the OD value at 610 nm with Microplate Reader

1.    For serum(plasma) and other liquid sample:

2.    For tissue/cell homogenate sample: