Hemoglobin Measurements - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 628

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
outfl ow 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 fl uid to a new tube.

1. Pipette 50 L calibrator into

wells of a clear bottom 96-well plate. Transfer 200

μ L distilled water (blank) and 50 μ

1.4. Procedures

μ L distilled

1.4.1. Procedures Using
a 96-Well Plate

water into the blank and calibrator wells.
2. Transfer 50 L 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).

μ L sample and 200 μ

1. Transfer 100 μ L calibrator and 1,000 μ L distilled water to a 1.4.2. Procedures Using

a Cuvette cuvette.

2. Transfer 100 μ L sample and 1,000 μ L reagent into a cuvette

and tap lightly to mix.
3. Put the cuvette into a spectrophotometer and read OD at
400 nm against water.

The concentrations of Hb in the samples are calculated using the
following equations:

1.5. Calculation

Hb levels in sample = (OD Sample − OD Water )/
(OD Calibrator − OD Water )× 100 × n (mg/dL)

OD Sample , OD Calibrator , and OD Water 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.