The purpose of this experiment is to test for the presence of water channels in RBC plasma membranes using the aquaprin inhibitor tetraethylammonium chloride (TEA).
1. Obtain 2 test tubes. Label one control. Label the other TEA. Transfer 5 ml 10% hct blood to each of the test tubes. Keep them both at room temperature.
2. Add 250 ul dH2O to the control blood and invert to mix 4X. Add 250 ul TEA to the TEA blood and invert to mix 4X. Incubate for 30 minutes at RT.
3. While the blood is incubating, gather 12 disposable spectrophotometer cuvettes and arrange them in four groups of three. These cuvettes will be used to assay the control blood. Gather 12 more disposable spectrophotometer cuvettes and arrange similarly in four groups of three. These cuvettes will be used to assay the TEA-treated blood.
4. Label the cuvettes according to the chart provided.
5. Using the blue automatic pipettor, transfer 1 ml of the appropriate hypotonic saline solution to each of the labeled test tubes according to the table provided.
6. When the 30-minute incubation period is over, prepare a control blank. To prepare this blank, add 1.0 ml of deionized water (0.00% saline) to a fresh cuvette. Next, add 100 µl of the control blood to the same cuvette. (100% of the cells added to the water will hemolyze). Invert 4X to mix. Later you will use this blank to calibrate the machine to 100 %T when assaying controls. Label it and set it aside until needed.
7. Next, prepare a TEA blank. To prepare this blank, add 1.0 ml of deionized water (0.00% saline) to a fresh cuvette. Next, add 100 µl of the TEA-treated blood to the same cuvette. (100% of the cells added to the water will hemolyze). Invert 4X to mix. Later you will use this blank to calibrate the machine to 100 %T when assaying TEA-treated blood. Label it and set it aside until needed.
When the 30-minute incubation time is complete and you have produced your blanks, perform the assays as described below.
a. Calibrate the spectrophotometer with the appropriate blank (either the control blank or the TEA blank).
b. Perform the following steps rapidly and carefully.
1. With a pipettor, add 100 µl of control or TEA-treated blood to one of the cuvettes containing one of the hypotonic salt solutions.
2. Immediately invert the cuvette 3X to mix. (Timer: start the timer at the third inversion.)
3. Immediately place the cuvette in the spectrophotometer and close the cuvette holder.
4. Record the %T of 625 nm light at 15-second intervals for 60 seconds.
| Cuvette | Solution (% NaCl) | 15 sec | 30 sec | 45 sec | 60 sec |
|---|---|---|---|---|---|
| A1 | 0.40% | 110.9 | 101.7 | 102 | 102.2 |
| A2 | 0.40% | 101.5 | 103 | 103.5 | 103.7 |
| A3 | 0.40% | 90.2 | 92.7 | 94.2 | 94.6 |
| B1 | 0.45% | 93.8 | 100.6 | 101.4 | 101.8 |
| B2 | 0.45% | 93.6 | 97.4 | 98.5 | 99.2 |
| B3 | 0.45% | 97.9 | 101.4 | 102.4 | 102.8 |
| C1 | 0.50% | 70.3 | 80.3 | 84.4 | 85.1 |
| C2 | 0.50% | 57.7 | 67.7 | 71.9 | 74.6 |
| C3 | 0.50% | 52.8 | 61.2 | 65.6 | 68.1 |
| D1 | 0.55% | 18.5 | 22.4 | 25.1 | 26.6 |
| D2 | 0.55% | 11.4 | 13 | 14.2 | 15 |
| D3 | 0.55% | 12.9 | 14.7 | 16 | 16.8 |
| Cuvette | Solution (% NaCl) | 15 sec | 30 sec | 45 sec | 60 sec |
|---|---|---|---|---|---|
| A1 | 0.40% | 97.8 | 101.4 | 102.3 | 102.7 |
| A2 | 0.40% | 98.6 | 101 | 101.8 | 102.2 |
| A3 | 0.40% | 96.4 | 100 | 101 | 106.4 |
| B1 | 0.45% | 31.5 | 39.6 | 44.4 | 47.3 |
| B2 | 0.45% | 37.6 | 43.2 | 47.3 | 50.1 |
| B3 | 0.45% | 39.9 | 52.3 | 57.6 | 61 |
| C1 | 0.50% | 17.5 | 20.2 | 21.7 | 23.2 |
| C2 | 0.50% | 13.4 | 15.5 | 16.9 | 17.8 |
| C3 | 0.50% | 20.4 | 23.1 | 24.9 | 26.3 |
| D1 | 0.55% | 6.5 | 6.9 | NaN | 7.4 |
| D2 | 0.55% | 5.9 | 6.2 | 6.4 | 6.5 |
| D3 | 0.55% | 5.1 | 5.4 | 5.5 | 5.6 |
| Group | Solution (% NaCl) | 15 sec | 30 sec | 45 sec | 60 sec |
|---|---|---|---|---|---|
| Control | 0.40% | 101 | 99 | 100 | 100 |
| Control | 0.45% | 95 | 100 | 101 | 101 |
| Control | 0.50% | 60 | 70 | 74 | 76 |
| Control | 0.55% | 14 | 17 | 18 | 19 |
| TEA-treated | 0.40% | 101 | 99 | 100 | 100 |
| TEA-treated | 0.45% | 95 | 100 | 101 | 101 |
| TEA-treated | 0.50% | 60 | 70 | 74 | 76 |
| TEA-treated | 0.55% | 14 | 17 | 18 | 19 |
Link to Lesson 1 | Link to Lesson 2
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