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
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
- 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
- Ouabain, bumetanide, and hydroxymethyl amiloride are prepared according to manufacturer instructions and stored < 2 wks at room temperature.
- Charybdotoxin is prepared according to manufacturer instructions and stored at -80°C.
- Ionophore A23187 is dissolved in EtOH.
Procedure: Erythrocyte preparation and hematology
- Blood is collected in K2-EDTA heparin tubes from isoflurane-anesthetized animals and is used within 1 h.
- Erythrocyte and reticulocyte counts are assessed with the ADVIA analyzer.
- Remaining whole blood is passed through cotton (to decrease the number of leukocytes) and centrifuged for 4 min at 4°C.
- Erythrocytes are washed four times with ice-cold CWS.
- 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.
- Reticulocyte counts and parameters are also assessed by Oxazine-750 staining on the ADVIA analyzer.
- Erythrocytes are washed four times with ice cold Mg-free choline chloride solution (Mg-free CWS).
- Erythrocyte are suspended at 50% with Mg-free CWS.
- An aliquot of the erythrocyte suspension is used for manual determination of hematocrit.
Procedure: Erythrocyte cation content
- 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).
- Samples are centrifuged at 300 rpm to pellet any membrane components and are stored at 4°C.
- Erythrocyte cation contents are determined by atomic absorption spectrophotometry.
Procedure: Erythrocyte gardos channel activity
- 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).
- Calcium ion concentration is calculated by using the dissociation constants for citrate and correcting for ionic strength and 0.15 mM MgCl2 presence.
- 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.
- Supernatants are aspirated, and tube tips containing the cell pellets are cut off.
- Erythrocyte-associated radioactivity is counted in a gamma-counter.
- Fluxes are calculated from linear regression slopes (potassium uptake is linear up to 5 min).
Procedure: Erythrocyte Na-K pump activity
- Freshly isolated erythrocytes (0.5 mL) suspended at 50% are added to 5 mL of NLS buffer and mixed carefully.
- Nystatin solution (40 mg/mL) is added while vortexing.
- 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.
- 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).
- Erythrocytes are washed five more times at 4°C in CWS to remove extracellular sodium or potassium contaminants.
- Washed erythrocytes are suspended to a hematocrit of 50%.
- Aliquots for ADVIA, ion content, and manual hematocrit are collected.
- Remaining cells (0.2 mL) are incubated with 8 mL flux media at 37°C in the presence or absence of 1 mM ouabain.
- 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.
- Supernatants are removed carefully and transferred to clean tubes for atomic absorption spectrophotometric determination of sodium ion content.
- 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).
- Na-K pump activity is estimated as the ouabain-sensitive fraction of sodium efflux into the supernatants.
Procedure: Erythrocyte Na-H exchanger activity
- Na-H exchanger activity is measured in nystatin-loaded cells as described above.
- Cells are placed into hypertonic flux media in the presence or absence of 10 µM hydroxymethyl amiloride (HMA).
- Na-H exchanger activity is estimated as the HMA-sensitive fraction of Na+ efflux.
Procedure: Erythrocyte K-Cl cotransporter activity
- Freshly isolated erythrocytes are suspended at 50% hematocrit after an aliquot is removed for ion content and ADVIA determinations as described above.
- Erythrocytes are incubated into either hypotonic NaCl medium or into isotonic NaCl medium at 37°C.
- Aliquots are removed after 5 and 25 min and immediately transferred to pre-cooled tubes.
- Flux is calculated from the slope of linear regression of K+ content vs. time.
- 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
- Reticulocytes (percent of RBC)
- Gardos channel activity
- Na-K pump activity
- Na-H exchanger activity
- K-Cl cotransporter activity