Isolation of alveolar type II cells by centrifugal elutriation

Summary Centrifugal elutriation (counterflow centrifugation) was used to develop a reproducible method for obtaining a nearly pure population of isolated alveolar type II cells. Lung was dissociated into individual cells with recrystallized trypsin, and the type II cells were partially purified by centrifugation on a discontinuous density gradient. The alveolar type II cells were finally purified by centrifugal elutriation. Cells were collected from the elutriator rotor by stepwise increases in flow rates. Cells obtained at flow rates of 7 and 14 ml per min were lymphocytes, other small cells, a few type II cells and cell debris; cells collected at flow rates of 18 and 22 ml per min were mainly type II cells; and cells collected at flow rates of 28, 34 and 43 ml per min were macrophages, some type II cells, other lung cells and cell aggregates. At flow rates of 18 and 22 ml per min, 1.9±1.0×10⁶ cells per rat lung (mean±S.D.,n=30) were recovered of which 86±6% were type II cells. At these flow rates, 94% of the cells excluded the vital dye erythrosin B from their cytoplasm. They consumed oxygen at a rate of 101±21 nmol per hr·10⁶ cells (mean±S.D.,n=4), and their oxygen consumption increased only 10% after 10mm sodium succinate was added. The cells incorporated [¹⁴C]leucine into protein and lipid for 4 hr. Electron micrographs of the cells collected at flow rates of 18 and 22 ml per min show a high percentage of morphologically intact alveolar type II cells. We conclude that centrifugal elutriation is a reproducible method for obtaining nearly pure, metabolically active alveolar type II cells. Postdoctoral trainee supported by Grants HL-05251 and HL-07192 from the National Heart, Lung and Blood Institute. This work was supported by U.S. Public Health Service Grants Program-Project HL-06285 and Pediatric Pulmonary SCOR HL-19185, and by a grant-in-aid from the American Heart Association (77-1098).     

Isolation of alveolar type II cells by centrifugal elutriation.

Centrifugal elutriation (counterflow centrifugation) was used to develop a reproducible method for obtaining a nearly pure population of isolated alveolar type II cells. Lung was dissociated into individual cells with recrystallized trypsin, and the type II cells were partially purified by centrifugation on a discontinuous density gradient. The alveolar type II cells were finally purified by centrifugal elutriation. Cells were collected from the elutriator rotor by stepwise increases in flow rates. Cells obtained at flow rates of 7 and 14 ml per min were lymphocytes, other small cells, a few type II cells and cell debris; cells collected at flow rates of 18 and 22 ml per min were mainly type II cells; and cells collected at flow rates of 28, 34 and 43 ml per min were macrophages, some type II cells, other lung cells and cell aggregates. At flow rates of 18 and 22 ml per min, 1.9 +/- 1.0 x 10(6) cells per rat lung (mean +/- S.D., n=30) were recovered of which 86 +/- 6% were type II cells. At these flow rates, 94% of the cells excluded the vital dye erythrosin B from their cytoplasm. They consumed oxygen at a rate of 101 +/- 21 nmol per hr . 10(6) cells (mean +/- S.D., n=4), and their oxygen consumption increased only 10% after 10 mM sodium succinate was added. The cells incorporated [14C]leucine into protein and lipid for 4 hr. Electron micrographs of the cells collected at flow rates of 18 and 22 ml per min show a high percentage of morphologically intact alveolar type II cells. We conclude that centrifugral elutriation is a reproducible method for obtaining nearly pure, metabolically active alveolar type II cells.     

Resolution of cells by centrifugal elutriation

Cell separation using the Beckman elutriator depends upon the flow rate of the medium and the centrifugal field employed. Changes in either the centrifugal field or the flow rate can be used to elute fractions of cells based on size. Even when these variables are held constant in the Beckman J21C centrifuge, a periodic pulse of cells is eluted. We have found that this anomolous elution is related to the temperature control system which gave a periodically pulsed temperature drop in the centrifuge well. The elution resulting from this change in temperature caused a shift in the modal cell size of the fraction eluted at a particular flow rate and centrifugal field. Because of this, the fractions have a larger size dispersion than fractions collected under conditions where refrigeration-related temperature fluctuations do not occur. We conclude that the temperature control system of the Beckman J21C centrifuge used with the Beckman elutriation rotor produces temperature fluctuations which prevent maximum resolution of cells.     

Isolation of cell cycle fractions by counterflow centrifugal elutriation

Counterflow centrifugal elutriation (CCE) has been used to fractionate cell populations on the basis of sedimentation properties, with minimal perturbation of metabolic function. Therefore, it is an ideal method for the isolation of cell cycle phase specific populations. We present modifications of the standard Beckman centrifugal elutriation system which permit standardization of the elutriation procedure and eliminate inter-run variability. We provide elutriation parameters for the cell cycle fractionation of a variety of cultured cell lines and suggest ways to improve the quality of the cell separations. In addition, we describe protocols for the fractionation of up to 3.50 X 10(8) cells in the small (JE-6B) Beckman elutriation system. This represents a four- to eight-fold increase in cell numbers over current cell fractionation procedures. Cell cycle populations containing greater than 95% G1, greater than 80% S, and greater than 70% G2/M were consistently obtained using these protocols. Finally, we analyzed phase-enriched fractions from several cultured cell lines for the cell cycle regulation of the enzyme thymidine kinase. The data confirm previous findings that CCE is an excellent means of obtaining physiologically unperturbed cell cycle phase specific fractions.     

Shrinkage of growing Escherichia coli cells by osmotic challenge.

The immediate response of growing Escherichia coli to changing external osmotic pressure was studied with stopped-flow turbidimetric measurements with a narrow-beam spectrophotometer. It is shown theoretically that in such a photometer rod-shaped bacteria have an apparent absorbance which is proportional to the inverse of the surface area. The apparent optical density, corrected for effects of alteration of the index of refraction of the medium, increased continuously as the external osmotic pressure was raised. Because of the short time scale of the measurements, the turbidity increases could result either from shrinkage of the cells or from plasmolysis, or both, but not from growth or metabolic adaptation. With low concentrations of pentose such that the external osmotic pressure was not greater than that inside the cells, plasmolysis would not occur and, consequently, only shrinkage of the previously stretched sacculus remains to account for the observed optical effects. Taking the osmotic pressure of the growing cells as 5 atmospheres (506 kPa), the turbidity changes correspond to the murein fabric having been stretched 20% beyond its unstressed equilibrium area during growth under the conditions used.     

Synchronization of 9L rat brain tumor cells by centrifugal elutriation

Asynchronous 9L cells were separated into relatively homogeneously-sized populations using centrifugal elutriation with both a conventional collection method and a long collection method. A substantial increase in the homogeneity of the volume distributions and in the degree of synchrony of the separated fractions was obtained using the long collection method. Autoradiographic data indicated that fractions containing ≥97% G₁ cells, ≥80% S cells, and 70–75% G₂ cells could be routinely recovered with this procedure. Recovery in these fractions varied from 5 to 8% of the total number of cells elutriated. The colony forming efficiency (CFE) of cells from fractions representing each phase of the cell cycle was a constant 60–70%, which was comparable to the 60–80% usually found for asynchronous 9L cells. The percentage of cells in the G₁, S, and G₂ phases in the elutriated fractions was more accurately determined from the volume distribution than from computer fits of the DNA histogram obtained from flow cytometry. In general, the degree of synchrony was related to the coefficient of variation (CV) of the volume distributions of the elutriated fractions. The CV was about 14% for all elutriated fractions. When the ≥97% G₁ population was allowed to progress to S and G₂, the CVs were about 17 and 20.2%, respectively. Thus, the best nonperturbing method for obtaining synchronous 9L cells in the S or G₂ phases was direct elutriation with the long collection method.     

Synchronization of 9L rat brain tumor cells by centrifugal elutriation

Asynchronous 9L cells were separated into relatively homogeneously-sized populations using centrifugal elutriation with both a conventional collection method and a long collection method. A substantial increase in the homogeneity of the volume distributions and in the degree of synchrony of the separated fractions was obtained using the long collection method. Autoradiographic data indicated that fractions containing greater than or equal to 97% G1 cells, greater than or equal to 80% S cells, and 70-75% G2 cells could be routinely recovered with this procedure. Recovery in these fractions varied from 5 to 8% of the total number of cells elutriated. The colony forming efficiency (CFE) of cells from fractions representing each phase of the cell cycle was a constant 60-70%, which was comparable to the 60-80% usually found for asynchronous 9L cells. The percentage of cells in the G1, S, and G2 phases in the elutriated fractions was more accurately determined from the volume distribution than from computer fits of the DNA histogram obtained from flow cytometry. In genereal, the degree of synchrony was related to the coefficient of variation (CV) of the volume distributions of the elutriated fractions. The CV was about 14% for all elutriated fractions. When the greater than or equal to 97% G1 population was allowed to progress to S and G2, the CVs were about 17 and 20.2%, respectively. Thus, the best nonperturbing method for obtaining synchronous 9L cells in the S or G2 phases was direct elutriation with the long collection method.     

Cell-cycle differences of HL-60 leukemia cells fractionated by centrifugal elutriation

HL-60 leukemia cells were fractionated into G1 and S/G2 populations using a rapid centrifugal elutriation technique, and studied for differences between the cell-cycle phases. The G1 fraction was found to contain smaller cells with a sedimentation velocity of 7 mm/h. The S/G2 fraction consisted of larger cells with a sedimentation velocity of 125 mm/h. The latter fraction was found to have a peak level of the enzyme (2'-5')An-binding protein, as compared to the G1 fraction, indicating a possible role for (2'-5')An-binding protein and its products in the growth regulation of these leukemic cells. In addition, cytofluorometric analysis of fractionated HL-60 cells indicates that elutriation is an effective fractionation method, rapidly yielding large numbers of cells for study, without the use of chemical treatments.     

Cell cycle synchronization of animal cells and nuclei by centrifugal elutriation

Synchronization of cells and nuclei is a powerful technique for the exact study of regulatory mechanisms and for understanding cell cycle events. Counterflow centrifugal elutriation is a biophysical cell separation technique in which cell size and sedimentation density differences of living cells are exploited to isolate subpopulations in various stages of cell cycle. Here, a protocol is described for the separation of phase-enriched subpopulations from exponentially growing Chinese hamster ovary cells at high-resolution power of elutriation. The efficiency of elutriation is confirmed by measuring the DNA content fluorimetrically and by flow cytometry. The resolution power of elutriation is demonstrated by the ability to fractionate nuclei of murine pre-B cells. The installation and elutriation by collecting 16-30 synchronized fractions, including particle size analysis, can be achieved in 4-5 h.     

Transmembrane glutathione cycling in growing Escherichia coli cells

Glutathione (GSH) plays an important role in bacterial cells, participating in maintenance of redox balance in the cytoplasm and in defense against many toxic compounds and stresses. In this study we demonstrate that in aerobic, exponentially growing Escherichia coli culture endogenous reduced glutathione undergoes continuous transmembrane cycling between the cells and medium. As a result of an establishment of a dynamic balance between GSH efflux and uptake, a constant extracellular concentration of GSH counting per biomass unit is maintained. The magnitude of this concentration strictly depends on external pH. GSH cycling is carried out in respiring cells and disturbed by influences, which change the level of ΔμH(+) and ATP. Export of GSH is modified by phosphate deficiency in the medium.     

Characterisation of hepatocyte sub-populations generated by centrifugal elutriation

We describe protocols for the fractionation of isolated hepatocytes into eight sub-populations using centrifugal elutriation. The distribution of fluorescein isothiocyanate and acridine orange in hepatocytes prepared from livers pre-perfused with one of these dyes is described and used as an indicator of acinar zone derivation for each population. The cytochrome P-450 content and response to induction by 3-methylcholanthrene and phenobarbitone; the distribution of lactate dehydrogenase, glucose-6-phosphatase, pyruvate kinase and tyrosine aminotransferase activities in the sub-populations is also reported. A marked asymmetry of distribution in all these activities was observed. On the basis of putative zone derivations (based on data of fluorescent dye distribution) of eight factors studied, the distributions of six were consistent with the sub-populations being derived from different acinar zones. Two major discrepancies were noted however, the distribution of pyruvate kinase activity and the response of the sub-populations to phenobarbitone. We conclude from this study that while a metabolic heterogeneity was revealed in the sub-populations generated, further characterisation is required to determine whether acinar zone separation has occurred and if so to what extent.     

Glucose transport of Escherichia coli growing in glucose-limited continuous culture.

Dilute cultures of wild-type Escherichia coli K12 and of derivatives impaired in one or other Enzyme-II component of the glucose phosphotransferase system were grown in continuous culture under glucose limitation. Cells harvested from the chemostat took up [U-14C]glucose from 0.1 mM solutions at rates directly related to the rates at which those cells had grown; the activity of the phosphotransferase system in those cells, rendered permeable with optimal accounts of toluene, parallels the ability of the cells to take up glucose. The capacity of these systems was rate-limiting for growth under the negligibly low glucose concentration in the chemostat, but was adequate to account for the stimulation of respiration observed when the cells were presented suddenly with excess glucose.     

Release of cell wall peptides into culture medium by exponentially growing Escherichia coli.

Escherichia coli W7 cells were found to release three different muropeptides into the culture medium: tetrapeptide (L-Ala-D-Glu-meso-diaminopimelic acid-D-Ala), tripeptide (L-Ala-D-Glu-meso-diaminopimelic acid), and a previously undescribed dipeptide (meso-diaminopimelic acid-D-Ala). From the rate of release of these three peptides, it was calculated that 6 to 8% of the murein in the sacculus was lost per generation.     

Separation of Kupffer and endothelial cells of the rat liver by centrifugal elutriation

Isolated non-parenchymal cells from rat liver were separated by centrifugal elutriation into two fractions consisting of structurally intact Kupffer and endothelial cells with purities of 91 and 95%, respectively. Purified Kupffer and endothelial cells showed nearly equal specific activities for the lysosomal enzyme acid phosphatase, whereas the specific activity of cathepsin D was about 3 times higher in Kupffer cells. It was calculated that a significant amount of the cathepsin D activity in the liver is present in the Kupffer cells.     

Intracellular protein breakdown in growing cells of Escherichia coli

1. When Escherichia coli was grown exponentially in a defined medium at 35 degrees , the rate of protein breakdown was initially rapid, but decreased to 0.6%/hr. after about 30min. The latter rate was maintained for at least 3.5hr. 2. The initial rapid rate may have been due to the presence of a small protein fraction (about 1%) that was degraded with a half-life of 13min. 3. The rate of protein degradation was the same during balanced growth at low rates imposed in a bactogen. However, it increased during the period immediately after a decrease of the growth rate.