Stress Response

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sample

5. The conjugate was prepared by adding 15uL of conjugate to 24mL of assay diluents prepared previously. Then diluted conjugate was immediately mixed and 200uL was pipette into each well using a multichannel pipette.

6. The plate was mixed by tapping for 5 minutes and the plate was incubated at room temperature for 55 minutes.

7. The plate was washed by pipetting 300 uL of 1x wash buffer and layers of tissue was gently tapped to remove solution and washing and cleaning was repeated at least 3 times.

8. 200uL of TMB solution was added to each well with a multichannel pipette.

9. Mixing was done by tapping and the plate was incubated in the dark at room temperature for 5 minutes.

10. Then 50uL of stop solution was added with a multichannel pipette

11. Bottom of the plate was wiped with water moistened, lint free cloth and dried

12. The plate was gone under reading process at 450nm.

(Process 10 to 12 was carried out within 10minutes.)

Results

Figure 1. Standard curve of cortisol level secretion measured via Salimetrics TM Salivary DHEA-S Kit with absorbance at 450m against concentration/ng/mL

Table 1. Absorbance at 450nm of average class data at 0, 5, 20, 60 and 120 minutes.

Stressor

Baseline(0min)

5min

20min

60min

120min

Physical (Cold/pain)

0.2932

0.3104

0.3188

0.3114

0.3153

Physical

(Exercise)

0.3084

0.3468

0.4024

0.3904

0.3967

Psychological(Mental)

0.3140

0.3267

0.4612

0.4008

0.4146

Control student

0.2136

0.2629

0.2765

0.2799

0.3299

From the equation derived from standard curve,

y = -0.089ln(x) + 0.1652

For absorbance of ‘Cold’ stress at baseline (0 min),

0.2932 = -0.089ln(x) + 0.1652

ln(x) = -1.4382

x = 0.2374 (Same calculations apply to all the absorbance values above)

Table 2. Average class data for cortisol concentration (ng/mL) at 0, 5, 20, 60 and 120 minutes.

Stressor

Baseline(0min)

5min

20min

60min

120min

Physical (Cold/pain)

0.2374

0.1956

0.1780

0.1935

0.1852

Physical

(Exercise)

0.2001

0.1300

0.0696

0.0796

0.0742

Psychological(Mental)

0.1879

0.1629

0.0359

0.0708

0.0607

Control student

0.5805

0.3336

0.2863

0.2756

0.1571

Figure 2.  Graph representing the mean ± standard error of the mean (SEM) of cortisol absorbance at 450nm at baseline (t=0 min), 5 and 20 min after 5 minutes of exercise, cold, singing and control.

Table 3. Average heart rate (mmHg) of class data at 0, 5 and 20 minutes.

Stressor

Baseline(0min)

5min

20min

Physical (Cold/pain)

78.38

78.71

77.38

Physical

(Exercise)

77.75

96.13

88.29

Psychological

(Mental)

87.21

90.88

84.13

Control

78.38

78.71

77.38

Figure 3. Graph representing the mean ± standard error of the mean (SEM) of heart rate (beats per minute) at baseline (t=0 min), 5 and 20 min after 5 minutes of exercise, cold, singing and control.

Table 4. Systolic and diastolic blood pressure (mmHg) of average class data at 0, 5 and 20 minutes.

Stressor

Baseline(0min)

5min

20min

Systolic