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PubMed2003

Assessment of sperm viability in boar, rabbit and rooster: a modification of the fluorometric ethidium bromide exclusion procedure.

The ethidium bromide (EtBr) exclusion procedure, a fluorometric method for measuring sperm cell viability, was studied to optimize the use of this technique on boar, rabbit and rooster semen. Diluted semen was used for boars and roosters. Diluted rabbit semen did not allow for reliable fluorescence readings; the interference of granules characteristic of rabbit seminal plasma was suggested as its cause. Therefore, rabbit semen was washed on several Percoll and Optiprep density gradients, with the aim of removing the granules from the sperm suspension. The complete absence of granules was not obtained, however, the best result was provided by the 35/70% Percoll density gradient. Most spermatozoa formed a loose pellet with low contamination. Although the washing procedure resulted in a selective action, Percoll washed semen was used to assess the EtBr procedure. The fluorescence intensities of stained fresh and stained digitonin-permeabilized samples were corrected, respectively, for the nonspecific fluorescence measures of fresh and digitonin-permeabilized samples both unstained. The contribution of the dye was subtracted from the corrected values, then the ratio between the corrected values of fresh and permeabilized cells provided the proportion of damaged cells in the sample. The working cell concentration range giving a constant proportion of damaged cells was set using diluted semen for boars and roosters (8-32 x 10(6) cell/ml) and Percoll washed semen for rabbits (4-16 x 10(6) cell/ml). The reliability of the fluorometric method was compared with the traditional nigrosin-eosin (NE) staining technique. The intactness of sperm samples containing known proportions of fresh and killed cells was measured in defined working cell ranges. For boars and roosters the values determined by fluorometry agreed closely with those determined using the NE method.

Animals↗
PubMed1978

Beta-blockade and blood-levels after low-dose oral propranolol: The hepatic "first-pass" threshold revisited.

Heart-rate, arterial pressure, and plasmarenin activity were determined in six normal subjects at rest and after an injection of 8 microgram isoprenaline with and without prior propranolol administered orally in a dose of 5 mg 8-hourly for a total of five doses. After propranolol, resting heart-rate, systolic pressure, and plasma-renin activity all fell significantly (P less than 0.05 to less than 0.001). When the isoprenaline-induced changes of heart-rate, diastolic pressure, and plasma renin activity without propranolol were compared to those with propranolol, these responses were greatly diminished (P less than 0.01 to less than 0.001). The percent blockade by propranolol of the isoprenaline-induced changes ranged from 65% for diastolic pressure to 77% for heart rate and 78% for plasma-renin activity. Propranolol levels determined by conventional fluorometry were below accurate detection limits, whereas those determined by gas-liquid chromatography ranged from 2.3 to 8.5 ng/ml. These findings, which demonstrate beta-blockade with low-dose propranolol, are not consistent with the existence of a postulated threshold for the hepatic "first-pass effect" in man, which is said to require saturation by single doses of 30 mg or more before propranolol enters the systemic circulation.

Administration, Oral↗
PubMed2001

Display of green fluorescent protein on Escherichia coli cell surface.

In this study, expression of green fluorescence protein (GFP) on the external surface of Escherichia coli was achieved by construction of a fusion protein between Lpp-OmpA hybrid and a GFP variant, GFPmut2. The GFP was fused in frame to the carboxyl-terminus of Lpp-OmpA fusion previously shown to direct various other heterologous proteins to E. coli cell surface. Western blot analysis of membrane fractions identified the Lpp-OmpA-GFP fusion protein with the expected size (43 kDa). Immunofluorescence microscopy, immunoelectron microscopy, protease and extracellular pH sensitivity assays further confirmed that GFP is anchored on the outer membrane. The GFP displayed on the E. coli outer surface retained its fluorescence and was not susceptible to the indigenous outer membrane protease OmpT even though there are two putative OmpT proteolytic sites present in GFP. Optimization of the expression conditions was conducted using fluorometry, eliminating cumbersome immuno-labeling procedures. Surface-displayed GFP could be used in a variety of applications including screening of polypeptide libraries, development of live vaccines, construction of whole cell allosteric biosensors, and signal transduction studies.

Journal Article↗
PubMed1996

Effects of diclofenac eye drops on corneal epithelial structure and function after small-incision cataract surgery.

PURPOSE: To investigate prospectively the effect of diclofenac sodium (DfNa) eye drops on corneal epithelial structure and function. METHODS: Seventeen patients with bilateral age-related cataract undergoing phacoemulsification combined with intraocular lens implantation were studied prospectively. After the surgery on both eyes, one eye each of these patients was assigned randomly to receive 0.1% DfNa eye drops three time daily (DfNa group), and the other eye served as control (control group). Vital stainings, tear function test, corneal sensitivity measurement, specular microscopy for corneal epithelium and endothelium, pachymetry, anterior fluorometry to measure epithelial barrier function, and laser flare-cell-metry were performed before and after surgery. RESULTS: There were no statistically significant differences between the DfNa and control groups in any measurement examined except for laser cell-flare-metry, in which the DfNa group demonstrated a significantly lower flare value at day 7 postoperatively (compare with the preoperative level of 73% +/- 41% in the DfNa group, and 130% +/- 98% in the control group, P=0.035). Epithelial cells in the DfNa group showed slight elongation and increased permeability postoperatively; however, there were no statistically significant differences with the control group. CONCLUSIONS: Administration of DfNa eye drops did not cause significant abnormalities in the corneal epithelial structure or function of patients who had undergone small-incision cataract surgery. Their safety for use in procedures such as excimer laser photorefractive keratectomy remains to be established.

Aged↗
PubMed2001

Extracts of ECL-cell granules/vesicles and of isolated ECL cells from rat oxyntic mucosa evoke a Ca2+ second messenger response in osteoblastic cells.

Surgical removal of the acid-producing part of the stomach (oxyntic mucosa) reduces bone mass through mechanisms not yet fully understood. The existence of an osteotropic hormone produced by the so-called ECL cells has been suggested. These cells, which are numerous in the oxyntic mucosa, operate under the control of circulating gastrin. Both gastrin and an extract of the oxyntic mucosa decrease blood calcium and stimulate Ca2+ uptake into bone. Conceivably, gastrin lowers blood calcium indirectly by releasing a hypothetical hormone from the ECL cells. The present study investigated, by means of fura-2 fluorometry, the effect of extracts of preparations enriched in ECL cell granules/vesicles from rat oxyntic mucosa on mobilization of intracellular Ca2+ in three osteoblast-like cell lines, UMR-106.01, MC3T3-E1 and Saos-2, and of extracts of isolated ECL cells in UMR-106.01 cells. The extracts were found to induce a dose-related rapid increase in intracellular Ca2+ concentrations in the osteoblast-like cells. The response was not due to histamine or pancreastatin, known ECL cell constituents, and could be abolished by pre-digesting the extracts with exo-aminopeptidase. The results show that the increase in [Ca2+](i) reflects a mobilization of Ca2+ from the endoplasmic reticulum. The observation of an increase in [Ca2+](i) also in murine embryonic fibroblasts show that the response is not limited to osteoblastic cells. The finding that the extracts evoked a typical Ca2+ -mediated second messenger response in osteoblastic cells provides evidence for the existence of a novel osteotropic peptide hormone (gastrocalcin), produced in the ECL cells, and supports the view that gastrectomy-induced osteopathy may reflect a lack of this hormone.

3T3 Cells↗
PubMed2002

High-molecular-weight protein hydrodynamics studied with a long-lifetime metal-ligand complex.

[Ru(2,2'-bipyridine)(2)(4,4'-dicarboxy-2,2'-bipyridine)](2+) (RuBDc) is a very photostable probe that possesses favorable photophysical properties including long lifetime, high quantum yield, large Stokes' shift, and highly polarized emission. In the present study, we demonstrated the usefulness of this probe for monitoring the rotational diffusion of high-molecular-weight (MW) proteins. Using frequency-domain fluorometry with a high-intensity, blue light-emitting diode (LED) as the modulated light source, we compared the intensity and anisotropy decays of RuBDc conjugated to immunoglobulin G (IgG) and immunoglobulin M (IgM), which show a six-fold difference in MW We obtained slightly longer lifetimes for IgM ( =428 ns in buffer) than IgG ( =422 ns in buffer) in the absence and presence of glycerol, suggesting somewhat more efficient shielding of RuBDc from water in IgM than in IgG. The anisotropy decay data showed longer rotational correlation times for IgM (1623 and 65.7 ns in buffer) as compared to IgG (264 and 42.5 ns in buffer). Importantly, the ratio of the long rotational correlation times of IgM to IgG in buffer was 6.2, which is very close to that of MW of IgM to IgG (6.0). The shorter correlation times are most likely to be associated with domain motions within the proteins. The anisotropy decays reflect both the molecular size and shape of the immunoglobulins, as well as the viscosity. These results show that RuBDc can have numerous applications in studies of high-MW protein hydrodynamics and in fluorescence polarization immunoassays (FPI) of high-MW analytes.

2,2'-Dipyridyl↗
PubMed1998

Apomyoglobin folding intermediates characterized by the hydrophobic fluorescent probe 8-anilino-1-naphthalene sulfonate.

Folding apomyoglobin intermediates were investigated by optical techniques including steady-state fluorescence, frequency domain fluorometry, and absorption spectroscopy. The investigated chromophores were the aromatic residues, i.e., tyrosyl and tryptophanyl residues, and the extrinsic probe (8-anilino-1-naphthalenesulfonate, ANS) which is particularly useful for studying partly structured forms appearing in the early stage of protein folding. The emission decay of the extrinsic probe as well as resonance energy transfer from tryptophanyl residues to ANS permitted to identify and characterize partly folded forms obtained under different experimental conditions. The results indicate that the intermediates so far detected (I-1 and I-2 states) are distinct structural states. The differences concern the solvent accessibility to the aromatic side chains and the conformational dynamics of the protein region forming the binding site for the extrinsic fluorophore.

Anilino Naphthalenesulfonates↗
PubMed2001

Establishment of variant PC12 subclones deficient in stimulation-secretion coupling.

Clonal rat pheochromocytoma (PC12) cells have been widely used to study the molecular mechanism of exocytosis. We have isolated variant PC12 subclones with deficiencies in stimulation-secretion coupling, by a single cell recloning, and investigated the defects. PC12-1G2 hardly released dopamine following high-K(+)-induced depolarization, but normal release was evoked by the Ca(2+)-ionophore, ionomycin. Fura-2 fluorometry indicated that a nicardipine-sensitive component of Ca(2+) influx was missing, suggesting that PC12-1G2 has defects in L-type Ca(2+) channel function. PC12-2B3 was not responsive to high-K(+)-induced depolarization and ionomycin, and voltage-dependent Ca(2+) entry was identical to that of the normal clone. Electron microscopy revealed that the number of vesicles adjacent or directly attached to the plasma membrane was decreased in PC12-2B3. The expression of presynaptic proteins was analyzed by immunoblotting using a panel of antibodies. Syntaxin 1, VAMP-2, SNAP-25, Munc18, Rab3C and Sec-6 were decreased compared to the control clone and that of synaptophysin was extremely low. PC12-D60 synthesized and released dopamine normally, but had almost lost its catecholamine-uptake activity. These results show that multiple PC12 cells variants are spontaneously generated, and that recloning can select PC12 subclones useful for the study of the molecular mechanisms of neurotransmitter release.

Amino Acid Sequence↗
PubMed2001

Osmotically induced cytosolic free Ca(2+) changes in human neutrophils.

Cytosolic free Ca(2+) concentration in neutrophils was measured by ratiometric fluorometry of intracellular fura2. Increasing the extracellular osmolarity, by either NaCl (300-600 mM) or sucrose (600-1200 mM), caused a rise in cytosolic free Ca(2+) (Delta(max) approximately equal to 600 nM). This was not due to cell lysis as the cytosolic free Ca(2+) concentration was reversed by restoration of isotonicity and a second rise in cytosolic free Ca(2+) could be provoked by repeating the change in extracellular osmolarity. Furthermore, the rise in cytosolic free Ca(2+) concentration occurred in the absence of extracellular Ca(2+), demonstrating that release of intracellular fura2 into the external medium did not occur. The osmotically-induced rise in cytosolic free Ca(2+) was not inhibited by either the phospholipase C-inhibitor U73122, or the microfilament inhibitor cytochalasin B, suggesting that neither signalling via inositol tris-phosphate or the cytoskeletal system were involved. However, the rise in cytosolic free Ca(2+) may have resulted from a reduction in neutrophil water volume in hyperosmotic conditions. As these rises in cytosolic Ca(2+) (Delta(max) approximately equal to 600 nM) were large enough to provoke changes in neutrophil activity, we propose that conditions which removes cell water may similarly elevate cytosolic free Ca(2+) to physiologically important levels.

Calcium↗
PubMed2002

Time-resolved fluorescence immunoassay (TRFIA) for the detection of Escherichia coli O157:H7 in apple cider.

An immunoassay based on immunomagnetic separation and time-resolved fluorometry was developed for the detection of E. coli O157:H7 in apple cider. The time-resolved fluorescent immunoassay (TRFIA) uses a polyclonal antibody bound to immunomagnetic beads as the capture antibody and the same antibody labeled with europium as the detection antibody. Cell suspensions of 10(1) to 10(8) E. coli O157:H7 and K-12 organisms per ml were used to test the sensitivity and specificity of the assay. The sensitivity of the assay was 10(3) E. coli O157:H7 cells with no cross-reaction with K-12. Pure cultures of E. coli O157:H7 (10(1) to 10(5) CFU/ml) in apple cider could be detected within 6 h, including 4 h for incubation in modified EC broth with novobiocin and 2 h for the immunoassay. When apple cider was spiked with 1 to 10(3) CFU/ml of E. coli O157:H7 and 10(6) CFU/ml of K-12, our data show that the high level of K-12 in apple cider did not impede the detection of low levels of O157:H7. The minimum detectable numbers of cells present in the initial inoculum were 10(2) and 10(1) CFU/ml after 4- and 6-h enrichment. The TRFIA provides a rapid and sensitive means of detecting E. coli O157:H7 in apple cider.

Antibodies, Bacterial↗
PubMed2000

Two methods for quantifying DNA extracted from poly(lactide-co-glycolide) microspheres.

DNA can be formulated with synthetic polymers such as poly(lactide-co-glycolide) (PLG) to generate microparticles. Researchers have used either UV spectroscopy or fluorometry with PicoGreen((R)) dye to quantify PLG-encapsulated DNA. While the sensitivity of DNA detection and quantification by PicoGreen is higher ( approximately 12 pg/ml) compared to UV ( approximately 0.5 microg/ml), each method as an analytical tool has limitations. The premise of this work addresses the usefulness and limitations of each method to determine encapsulation efficiencies in PLG microspheres post-process, and to quantify release of DNA from microspheres during in vitro release experiments. In addition, assay conditions for accurate and reproducible extraction of DNA from PLG microspheres using a biphasic (aqueous/organic) solvent system are described. It was also determined that residual poly(vinyl alcohol) and DNA isoforms (linear, nicked, supercoiled) affected PicoGreen/DNA fluorescence values.

DNA↗
PubMed1986

Chenodeoxycholic acid therapy in erythrohepatic protoporphyria.

The short-term effect of chenodeoxycholic acid administration on the excretion of protoporphyrin was investigated in 5 patients suffering from erythrohepatic protoporphyria. Faeces were collected for 7 days, 10 ml of bile was sampled daily and blood was drawn every 2 to 3 days. Chenodeoxycholic acid was given in a dosage of 15 mg/kg/day from the 8th day. Collection of faeces, bile and blood was then continued for 10 more days. Protoporphyrin concentration was measured by high-pressure liquid chromatography and fluorometry. Following the administration of chenodeoxycholic acid the concentration of protoporphyrin in faeces and bile decreased significantly. In addition, all patients showed a significant decrease in erythrocyte protoporphyrin concentration. These results indicate that chenodeoxycholic acid therapy causes a marked decrease in the excretion of protoporphyrin in patients with erythrohepatic protoporphyria. The subsequent decrease in erythrocyte protoporphyrin suggests that chenodeoxycholic acid inhibits the production of protoporphyrin in the liver.

Chenodeoxycholic Acid↗
PubMed1999

Epidermal growth factor- and hepatocyte growth factor-receptor activity in serum-free cultures of human hepatocytes.

BACKGROUND/AIMS: Serum-free primary cultures of hepatocytes are a useful tool to study factors triggering hepatocyte proliferation and regeneration. We have developed a chemically defined serum-free system that allows human hepatocyte proliferation in the presence of epidermal growth factor and hepatocyte growth factor. METHODS: DNA synthesis and accumulation were determined by [3H]thymidine incorporation and fluorometry, respectively. Western blot analyses and co-immunoprecipitations were used to investigate the association of proteins involved in epidermal growth factor and hepatocyte growth factor activation and signaling: epidermal growth factor receptor, hepatocyte growth factor receptor (MET), urokinase-type plasminogen activator and its receptor, and a member of the signal transducer and activator of transcription family, STAT-3. RESULTS: Primary human hepatocytes proliferated under serum-free conditions in a chemically defined medium for up to 12 days. Epidermal growth factor-receptor and MET were present and functional, decreasing over time. MET, urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor co-precipitated to varying degrees during the culture period. STAT-3 co-precipitated with epidermal growth factor-receptor and MET to varying degrees. CONCLUSIONS: Proliferation of human hepatocytes can improve by modification of a chemically defined medium originally used for rat hepatocyte cultures. In these long-term cultures of human hepatocytes, hepatocyte growth factor and epidermal growth factor can stimulate growth and differentiation by interacting with their receptors and initiating downstream signaling. This involves complex formation of the receptors with other plasma membrane components for MET (urokinase-type plasminogen activator in context of its receptor) and activation of STAT-3 for both receptors.

Adolescent↗
PubMed1997

Inducible expression of N-methyl-D-aspartate (NMDA) receptor channels from cloned cDNAs in CHO cells.

To develop a drug screening system, we introduced expression vectors carrying the mouse N-methyl-D-aspartate (NMDA) receptor channel epsilon1 and zeta1 subunit cDNAs under the promoter of the Drosophila heat shock protein hsp70 into Chinese hamster ovary (CHO) cells. We selected clonal cell lines by means of RNA blot hybridization and fura-2 fluorometry. One of these cell lines, ZE1-1, optimally expressed the epsilon1 and zeta1 subunit mRNAs when induced by an incubation at 43 degrees C for 2 h. Heated ZE1-1 cells exhibited the NMDA-induced intracellular Ca2+ elevation, whereas unheated they showed no such response. NMDA and L-glutamate, but not alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and kainate, induced an increase in the intracellular Ca2+ concentration. The response to the agonists was marginal in the absence of glycine, and diminished by Mg2+ and NMDA receptor antagonists. Furthermore, exposure to agonists of ZE1-1 cells expressing the epsilon1/zeta1 NMDA receptor channel resulted in the release of lactate dehydrogenase (LDH) activity in the culture medium indicating agonist-induced cell death. NMDA receptor antagonists inhibited the LDH activity release. These results suggest that ZE1-1 cells will provide a useful screening system for novel drugs acting on the epsilon1/zeta1 NMDA receptor channel.

Animals↗
PubMed2002

Metal-induced tolerance in the freshwater microbenthic diatom Gomphonema parvulum.

The benthic diatom Gomphonema parvulum Kützing is a common species in both clean and metal contaminated rivers. Our aim was to investigate whether metal-induced tolerance could explain the persistance of this taxon under metal polluted conditions. G. parvulum strains were isolated from a Zn- and Cd-contaminated stream and from a relatively clean ("reference") stream. The strains were cultured in synthetic medium as mono-specific biofilms to maintain their specific benthic growth features. Moreover, the strain from the metal polluted stream was cultured in plain and Zn- and Cd-enriched synthetic medium. Short-term (5 h) toxicity experiments with Zn were performed with the strains using pulse amplitude modulated (PAM) fluorometry. Zn lowered significantly the minimal chlorophyll fluorescence (F0) and the photon yield (phi(p)) of the exposed strains after 5 h exposure. The actual Zn concentrations that caused a 50% reduction (EC50's) of the phi(p) of the strain from the metal polluted stream were significantly higher than those of the isolate from the unpolluted stream. The absence of tolerance to Cu of the "polluted" strain indicated that Zn tolerance resulted from specific induction by chronic exposure to Zn in the field. Observations on field biofilms confirmed a higher tolerance of the G. parvulum population from the polluted stream than of the G. parvulum population from the reference stream. A genetic nature of this metal adaptation was supported by the persistance of the Zn tolerance of the polluted strain 2 years after isolation.

Adaptation, Physiological↗
PubMed1997

Fumonisins as a possible contributory risk factor for primary liver cancer: a 3-year study of corn harvested in Haimen, China, by HPLC and ELISA.

Employing HPLC fluorometry, gas-liquid chromatography (GLC) and a novel enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody, 40 corn samples, each collected in 1993 from agricultural stocks for human consumption in Haimen (Jiangsu County) and Penlai (Shandong Province), high- and low-risk areas for primary liver cancer (PLC) in China, respectively, were analysed for fumonisins (FBs), aflatoxins (AFs) and trichothecenes. Levels and positive rates of FBs and deoxynivalenol (DON) were significantly higher in Haimen than in Penlai. ELISA of the 40 corn samples harvested in the two areas in 1994 revealed that FB contamination levels and rates in these areas were comparable to those observed in 1993 in Haimen. ELISA analysis of 1993 and 1994 products revealed a wide occurrence of AFB1 but the positive rates as well as levels were not significantly different between these areas. ELISA of the same sample number of corn harvested in 1995 revealed that FB contamination in Haimen was significantly higher than in Penlai. These 3-yearly surveys of corn samples (240 in total) demonstrated that corn harvested in Haimen was highly contaminated with FBs and that the contamination level, as well as positive rate in 1993 and 1995, were 10-50-fold higher than those in Penlai, suggesting FBs as a risk factor for promotion of PLC in endemic areas, along with the trichothecene DON. Co-contamination with AFs, potent hepatocarcinogens, was assumed to play an important role in the initiation of hepatocarcinogenesis.

Aflatoxins↗
PubMed1998

A 4-year study of plasma ochratoxin A in a selected population in Tokyo by immunoassay and immunoaffinity column-linked HPLC.

Employing a competitive ELISA (cELISA) based on monoclonal antibody and a novel immunoaffinity-column (IAC)-linked HPLC-fluorometry, ochratoxin A (OTA) levels in the plasma of 184 healthy volunteers (130 males, 54 females) were surveyed in Tokyo during the 4 years from 1992 to 1996. It was found that 85% of the cases were positive for OTA except 38% in 1994, and an average value in the positives was estimated as 68 pg/ml. This suggests that the population in Tokyo is exposed to OTA at high frequency, although the level in plasma is far less than that reported in Europe and Canada. A partial contribution of fermented dietary foodstuffs, coffee and wine is suspected as the source of the OTA observed in the human plasma.

Chromatography, Affinity↗
PubMed2002

Granulocytes from patients with paroxysmal nocturnal hemoglobinuria and normal individuals have the same sensitivity to spontaneous apoptosis.

OBJECTIVE: The aim of this study was to determine whether granulocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) are more or less intrinsically sensitive to spontaneous apoptosis than granulocytes from healthy individuals. Resistance to apoptosis has been suggested as an explanation for the proliferation or selection of PNH clones. PATIENTS AND METHODS: Peripheral blood granulocytes from five patients with PNH, five patients with myelodysplastic syndrome (MDS), and five healthy volunteers were cultured in the absence of serum. Spontaneous apoptosis of the granulocytes was assessed every 6 hours by flow cytometry. The expression levels of CD16b, CD95, and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor also were studied by flow cytometry, and caspase-3 activity was measured by fluorometry. RESULTS: There were no significant differences in the proportion or absolute numbers of apoptotic and apoptotic/dead granulocytes between the cells from PNH patients and healthy individuals, whereas those from MDS patients showed significantly lower frequencies of apoptotic granulocytes compared with normal controls. The proportion of CD16b(-) granulocytes was not significantly different among the three groups during in vitro culture. CD95 and GM-CSF receptor was not significantly increased in cultured granulocytes or noncultured granulocytes from, respectively, patients with PNH and normal controls. Caspase-3 activity significantly decreased in cultured granulocytes from MDS patients, but not in granulocytes from PNH patients. CONCLUSIONS: Granulocytes from PNH patients did not display a reduced sensitivity to spontaneous apoptosis, suggesting that the apoptosis of blood cells in PNH may not be an important factor in proliferation or selection of PNH clones. These findings are in agreement with the normal lifespan of granulocytes in vivo.

Adult↗