Competition Experiment Protocol
Liao NO Group
2001/07/25
HEPES Buffer Preparation
Description:
Protocol for preparation of 40 mM HEPES with 5 mM Glucose. NaCl is added to adjust osmolarity to ~285 mOsm
Reagents:
Milli-Q filtered water
9.52 g/L HEPES (SigmaUltra)
0.90 g/L Glucose (Dextrose)
7.00 g/L NaCl
Procedure:
1. Add reagents to a sterile container of appropriate volume.
2. Add required volume of Milli-Q filtered water.
3. Add a magnetic stir-bar to buffer solution and mix using a stirrer.
4. When all reagents have dissolved, check osmolarity.
5. After calibrating the pH meter, adjust pH to 7.40 by the addition of concentrated NaOH (Increases pH) or HCl (Decreases pH).
6. Sterilize buffer solution by filtration.
7. Check osmolarity again.
8. Label buffer solution with: buffer name, pH, osmolarity, date, initials.
Red Blood Cell Preparation
Description:
Protocol for preparation of purified red blood cells (RBCs). Fresh blood must be used else lysis may be a problem.
Procedure:
1. Prepare a 50/50 mixture by mass of SigmaCell Type 50 Cellulose (S-5504) and alpha-cellulose (C-8002) in a beaker 4 times the volume of the total mixture added.
2. Cap the beaker with aluminum foil and autoclave the cellulose mixture.
3. Add HEPES buffer (~2-3x the celluose volume) to the autoclaved cellulose mixture to make a slurry.
4. Remove the membrane from a bottle top filter unit (Corning 430624) and replace with an autoclaved Type 1 Whatman paper cut to the appropriate size. Be sure that conditions and instruments used are sterile.
5. Attach filter unit to a 50 mL centrifuge tube (Fisher 05-539-10).
6. Pour cellulose mixture into filter unit until the packed column volume is twice the volume of blood to be filtered.
7. Obtain blood in an anticoagulant tube. Typically 10 mL of whole blood is needed per sample.
8. Centrifuge blood at 800 g and aspire and discard the supernatant and buffy coat.
9. Replace aspired volume with equivolume of HEPES buffer. Gently mix.
10. Gently pour blood onto cellulose column (being sure not to disturb the column).
11. Add another volume of HEPES buffer to elute RBCs.
12. Centrifuge purified RBCs at 800 g and aspire and discard the supernatant.
13. Repeat step 12. until the supernatant is clear. Typically three total wash steps are required.
Competition Experiment
Description:
Protocol for competition experiment.
Procedure:
1. Obtain washed and purified red blood cells (RBCs). Typically 1 mL of packed RBCs is used for each sample.
2. Prepare pretreated RBCs as needed.
3. For the first trial, at least 5 samples should be prepared:
a. oxyHb (monitor auto-oxidation)
b. oxyHb + NO donor (monitor NO released by donor)
c. oxyHb + RBC + NO donor (monitor NO uptake by untreated RBC)
d. oxyHb/metHb mixture + treated RBC (monitor oxidation/reduction by treated RBC)
e. oxyHb + treated RBC + NO donor (determine NO uptake by treated RBC)
4. Prepare a 10 µM oxyhemoglobin (oxyHb) stock solution in the 40 mM HEPES buffer.
5. Dilute RBCs to 15% hematocrit using the 10 µM oxyHb solution.
6. Use a volume, equivolume to the sample, of the 10 µM oxyHb as a control.
7. If needed, prepare a solution of 5 µM oxyHb and 5 µM metHb to monitor oxidation/reduction due to RBC treatments.
8. Add 15 µM Spermine/NONOate and gently mix samples.
9. Start timer.
10. Mix samples again and transfer each sample to its own 20 mL syringe.
11. Place syringes on a rotating device to maintain cell suspension.
12. Every 20 minutes, extrude a 1 mL aliquot of each sample into separate sterile 1.7 mL microcentrifuge tube.
13. Centrifuge at 16,000 g for 15 seconds.
14. Pipet out the supernatant and measure the absorbance over the range: 380 - 700 nm.
15. Save spectra and repeat measurement 3 or more times (Spectra names must follow the format outlined by the program).
16. Measure hematocrit of each RBC containing sample.
17. After data is collected, run program to determine NO uptake rate.