Project protocol - Contents

• Workflow and sampling
• Equipment and supplies
• Reagents and solutions
• Procedure: Drug and chemical preparation
• Procedure: Erythrocyte preparation and hematology
• Procedure: Erythrocyte cation content
• Procedure: Erythrocyte gardos channel activity
• Procedure: Erythrocyte Na-K pump activity
• Procedure: Erythrocyte Na-H exchanger activity
• Procedure: Erythrocyte K-Cl cotransporter activity
• Definitions and calculations
• Data

Workflow and sampling

Step	Procedure	Equipment	Data collected
1	Drug and chemical preparation	-	-
2	Erythrocyte preparation	-	-
3	Hematology	ADVIA	MCH, CHCM, MCH, % reticulocytes
4	Cation content	Spectrophotometer	Erythrocyte Na+, K+, Mg2+
5	Ion transport activity	Gamma-counter, spectrophotometer	Erythrocyte gardos channel activity, Na-K pump activity, Na-H exchanger activity, K-Cl cotransporter activity

Equipment and supplies

• ADVIA 120 hematology analyzer
• Atomic absorption spectrophotometer (AAnalyst 800, Perkin Elmer, Norwalk CT)
• Gamma-counter (Model 41600 HE, Isomedic ICN Biomedicals, Costa Mesa CA)
• K2-EDTA heparin tubes for blood collection
• Sorvall Legend RT centrifuge (Thermo Scientific, Asheville NC)
• Software program for mouse blood hematology (Siemens Diagnostic Solutions, Tarrytown NY)

Reagents and solutions

• Charybdotoxin (ChTX; Sigma, St. Louis MO)
• Ouabain (Sigma, St. Louis MO)
• Bumetanide (Sigma, St. Louis MO)
• Hydroxymethyl amiloride (HMA; Sigma, St. Louis MO)
• Ionophore A23187 (Calbiochem-Novabiochem, La Jolla CA)
• ⁸⁶Rb (Du Pont-New England Nuclear)
• Oxazine 750 (Sigma, St. Louis MO)
• Acationox (Sherwood Medical Industries, Inc)
• Physiological saline
• Isoflurane
• Nystatin
• Choline washing solution (CWS; 172 mM choline chloride, 1 mM MgCl₂, 10 mM Tris-MOPS pH 7.4)
• Choline washing solution magnesium-free (Mg-free CWS; 172 mM choline chloride, 10 mM Tris-MOPS pH 7.4, 10 mM sucrose)
• Influx media (165 mM NaCl, 2 mM KCl, 0.15 mM MgCl₂, 1 mM ouabain, 10 mM Tris-MOPS pH 7.4, 10 µM bumetanide, 10 µCi/L ⁸⁶Rb)
• Flux media (155 mM choline chloride, 10 mM KCl, 1 mM MgCl₂, 10 mM glucose, 10 mM Tris-MOPS pH 7.4)
• Hypertonic flux media (150 mM choline chloride, 200 mM sucrose, 1 mM MgCl₂, 10 mM glucose, 1 mM ouabain, 0.01 mM bumetanide, 10 mM Tris-MOPS pH 7.4)
• NLS buffer (77 mM NaCl, 77 mM KCl, 55 mM sucrose)
• NLS-BSA buffer (77 mM NaCl, 77 mM KCl, 55 mM sucrose, 0.5 mM KPO₄, 0.5 mM NaPO₄, 10 mM glucose, 0.1% bovine serum albumin (fraction V))
• Hypotonic NaCl medium (115 mM NaCl, 1 mM MgCl₂, 10 mM glucose, 10 mM Tris-MOPS pH 7.4, 1 mM ouabain, 0.01 bumetanide)
• Isotonic NaCl medium (160 mM NaCl, 1 mM MgCl₂, 10 mM glucose, 10 mM Tris-MOPS pH 7.4)
• Citrate buffer (1 mM pH 7.4)
• Ethylene glycol tetraacetic acid (EGTA)
• n-butyl phthalate

Procedure: Drug and chemical preparation

1. Ouabain, bumetanide, and hydroxymethyl amiloride are prepared according to manufacturer instructions and stored < 2 wks at room temperature.
2. Charybdotoxin is prepared according to manufacturer instructions and stored at -80°C.
3. Ionophore A23187 is dissolved in EtOH.

Procedure: Erythrocyte preparation and hematology

1. Blood is collected in K2-EDTA heparin tubes from isoflurane-anesthetized animals and is used within 1 h.
2. Erythrocyte and reticulocyte counts are assessed with the ADVIA analyzer.
3. Remaining whole blood is passed through cotton (to decrease the number of leukocytes) and centrifuged for 4 min at 4°C.
4. Erythrocytes are washed four times with ice-cold CWS.
5. An aliquot of 30 µL is diluted up to 300 µL with physiological saline and run on the ADVIA analyzer to obtain RBC index values.
6. Reticulocyte counts and parameters are also assessed by Oxazine-750 staining on the ADVIA analyzer.
7. Erythrocytes are washed four times with ice cold Mg-free choline chloride solution (Mg-free CWS).
8. Erythrocyte are suspended at 50% with Mg-free CWS.
9. An aliquot of the erythrocyte suspension is used for manual determination of hematocrit.

Procedure: Erythrocyte cation content

1. Cells are diluted in 0.02% acationox (1:50 for sodium and magnesium; 1:25 for calcium) or in double-distilled water (1:500 for potassium).
2. Samples are centrifuged at 300 rpm to pellet any membrane components and are stored at 4°C.
3. Erythrocyte cation contents are determined by atomic absorption spectrophotometry.

Procedure: Erythrocyte gardos channel activity

1. Erythrocytes are suspended at a hematocrit of 2% (hematocrit determination described above) in influx media in the presence or absence of 50 nM Charybdotoxin (free ionic calcium in the influx media is buffered to 7 µM with citrate buffer).
2. Calcium ion concentration is calculated by using the dissociation constants for citrate and correcting for ionic strength and 0.15 mM MgCl₂ presence.
3. At time 0 min, A23187 ionophore (5 µM) is added, and aliquots at 2 and 5 min are removed and immediately spun down through 0.8 mL of cold media containing 5 mM EGTA buffer and an underlying cushion of n-butyl phthalate.
4. Supernatants are aspirated, and tube tips containing the cell pellets are cut off.
5. Erythrocyte-associated radioactivity is counted in a gamma-counter.
6. Fluxes are calculated from linear regression slopes (potassium uptake is linear up to 5 min).

Procedure: Erythrocyte Na-K pump activity

1. Freshly isolated erythrocytes (0.5 mL) suspended at 50% are added to 5 mL of NLS buffer and mixed carefully.
2. Nystatin solution (40 mg/mL) is added while vortexing.
3. Erythrocyte suspension is incubated 20 min on ice (mixed every 5 min) and then centrifuged at 4°C and incubated another 20 min on ice in the same solution without nystatin.
4. Nystatin-loaded cells are washed four times in NLS-BSA buffer with an incubation of 10 min at 37°C between washes (with gentle shaking).
5. Erythrocytes are washed five more times at 4°C in CWS to remove extracellular sodium or potassium contaminants.
6. Washed erythrocytes are suspended to a hematocrit of 50%.
7. Aliquots for ADVIA, ion content, and manual hematocrit are collected.
8. Remaining cells (0.2 mL) are incubated with 8 mL flux media at 37°C in the presence or absence of 1 mM ouabain.
9. At time points (5 and 25 min) aliquots are removed (1.3 mL) in triplicate and transferred to pre-cooled tubes and centrifuged at 4°C.
10. Supernatants are removed carefully and transferred to clean tubes for atomic absorption spectrophotometric determination of sodium ion content.
11. Fluxes are calculated from linear regression slopes in the presence and absence of ouabain added to the flux media (there is approximately 5-10 min incubation time of the cells in the presence of ouabain before the beginning of the flux assay).
12. Na-K pump activity is estimated as the ouabain-sensitive fraction of sodium efflux into the supernatants.

Procedure: Erythrocyte Na-H exchanger activity

1. Na-H exchanger activity is measured in nystatin-loaded cells as described above.
2. Cells are placed into hypertonic flux media in the presence or absence of 10 µM hydroxymethyl amiloride (HMA).
3. Na-H exchanger activity is estimated as the HMA-sensitive fraction of Na⁺ efflux.

Procedure: Erythrocyte K-Cl cotransporter activity

1. Freshly isolated erythrocytes are suspended at 50% hematocrit after an aliquot is removed for ion content and ADVIA determinations as described above.
2. Erythrocytes are incubated into either hypotonic NaCl medium or into isotonic NaCl medium at 37°C.
3. Aliquots are removed after 5 and 25 min and immediately transferred to pre-cooled tubes.
4. Flux is calculated from the slope of linear regression of K⁺ content vs. time.
5. K-Cl cotransporter activity is estimated by subtracting total K⁺ efflux into hypotonic NaCl medium from the isotonic medium and expressed as volume-dependent K⁺ efflux.

Definitions and calculations

• MCV: mean RBC corpuscular volume
• CHCM: RBC corpuscular hemoglobin concentration mean
• MCH: mean RBC corpuscular hemoglobin content

Data collected by investigator

• MCH
• CHCM
• MCH
• Reticulocytes (percent of RBC)
• Na⁺
• K⁺
• Mg²⁺
• Gardos channel activity
• Na-K pump activity
• Na-H exchanger activity
• K-Cl cotransporter activity