Fig. 1.
Photograph of the model 6505 UV/vis spectrophotometer from Jenway.
4.
Cuvettes.
5.
Microplate reader.
Fig. 2.
Photograph of the Bio-Tek EL800 microplate reader.
6.
QuantiChrom™ Hb Assay Kit (BioAssay Systems). Reagent and Calibrator are included in the kit.
7.
Pipetting devices and Pipet Tips.
8.
0.1 mol/L PBS.
9.
Tubes.
10.
Distilled water.
11.
Centrifuge.
12.
Clear bottom 96-well plate.
1.2 Kit Contents
1.
50 mL Reagent
2.
10 mL Calibrator
1.3 Sample Preparation
1.
Inject sodium pentobarbital (50 mg/kg, i.p.) to anesthetize the Sprague–Dawley (SD) rats (250–300 g).
2.
Perfuse the rats with 0.1 mol/L PBS intracardially until the outflow from the right atrium is colorless.
3.
Remove the brains rapidly and put them into preweighed tubes.
4.
Weight the tissues on an electronic balance.
5.
Add 0.1 mol/L PBS into the tubes and homogenize the tissues.
6.
Centrifuge the homogenized tissues for 30 min at a speed of 13,000 × g, and then transfer the supernatant fluid to a new tube.
1.4 Procedures
1.4.1 Procedures Using a 96-Well Plate
1.
Pipette 50 μL distilled water (blank) and 50 μL calibrator into wells of a clear bottom 96-well plate. Transfer 200 μL distilled water into the blank and calibrator wells.
2.
Transfer 50 μL sample and 200 μL reagent into a well and tap plate lightly to mix. Avoid bubble formation during the pipetting steps.
3.
Incubate for 5 min at room temperature.
4.
Put the plate into a microplate reader and read OD at 395–405 nm (peak 400 nm).
1.4.2 Procedures Using a Cuvette
1.
Transfer 100 μL calibrator and 1,000 μL distilled water to a cuvette.
2.
Transfer 100 μL sample and 1,000 μL reagent into a cuvette and tap lightly to mix.
3.
Put the cuvette into a spectrophotometer and read OD at 400 nm against water.
1.5 Calculation
The concentrations of Hb in the samples are calculated using the following equations:
Hb levels in sample = (ODSample − ODWater)/(ODCalibrator − ODWater)× 100 × n (mg/dL)
ODSample, ODCalibrator, and ODWater are OD values of the samples, the calibrator, and water. 100 mg/dL is the equivalent hemoglobin concentration of the diluted calibrator. n is the dilution factor.
1.6 Advantages and Limitations
1.6.1 Advantages
1.
Detects Hb in multiple species, including rat, mouse, pig, and human.
2.
Measures Hb levels in brain, serum, plasma, and blood.
4.
Assays can be executed in a 96-well plate or a cuvette.
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