Separate actions of different colony stimulating factors from human placental conditioned medium on human hemopoietic progenitor cell survival and proliferation

More than 20% of human granulocyte-macrophage and eosinophil colony-forming cells survived in agar culture for up to 4 days without the addition of exogenous colony stimulating factors (human placental-conditioned medium, HPCM). Survival was reduced slightly but not significantly, by the removal of adherent cell populations. Significant survival occurred even when only 100 cells enriched for colony-forming cells (CFCs) were cultured per dish. When individual colonies, initiated by stimulation with HPCM for 5 days, were transferred to dishes without HPCM, subsequent proliferation was significantly reduced compared with control cultures containing HPCM. Using the fluorescence-activated cell sorter and the fluoresceinated lectin from Lotus tetragonolobus, two populations of marrow cells were obtained, one enriched for day 7 and the other for day 14 colony-forming cells. Two colony-stimulating factors fractionated from HPLCM (CSF^β and CSF^α) have been shown previously to stimulate the day 7 and day 14 colony-forming cell populations, respectively. Developing clones from cultures initiated with CSFβ died between the fifth and tenth day of culture after transfer to dishes with CSFα or CSFβ or to dishes with no stimulus. Cells in clusters initiated with CSFα proliferated significantly between the fifth and tenth day of culture when transfered to CSFα or CSFβ but not when transfered to dishes with not stimulus. These studies provide further evidence for the existence of two subtypes of human granulocyte-macrophage progenitor cells each under the primary control of a specific regulator and indicate that these two regulators can both act on some developing clones of cells.     

Safe sorting of GFP-transduced live cells for subsequent culture using a modified FACS vantage.

BACKGROUND: A stream-in-air cell sorter enables rapid sorting to a high purity, but it is not well suited for sorting of infectious material due to the risk of airborne spread to the surroundings. METHODS: A FACS Vantage cell sorter was modified for safe use with potentially HIV infected cells. Safety tests with bacteriophages were performed to evaluate the potential spread of biologically active material during cell sorting. Cells transduced with a retroviral vector carrying the gene for GFP were sorted on the basis of their GFP fluorescence, and GFP expression was followed during subsequent culture. RESULTS: The bacteriophage sorting showed that the biologically active material was confined to the sorting chamber. A failure mode simulating a nozzle blockage resulted in detectable droplets inside the sorting chamber, but no droplets could be detected when an additional air suction from the sorting chamber had been put on. The GFP transduced cells were sorted to 99% purity. Cells not expressing GFP at the time of sorting did not turn on the gene during subsequent culture. Un-sorted cells and cells sorted to be positive for GFP showed a decrease in the fraction of GFP positive cells during culture. CONCLUSIONS: Sorting of live infected cells can be performed safely and with no deleterious effects on vector expression using the modified FACS Vantage instrument.     

Influx流式仪分选肺腺癌A549细胞SP亚群的条件优化

目的：通过分选肺腺癌A549细胞的侧群细胞（side population cells,SP）,了解影响Influx分选SP的各种因素,为成功分选其他肿瘤细胞的SP亚群提供方法参考.方法：（1）细胞的制备：取对数生长期的细胞,制备单细胞悬液,用Hoechst33342及PI标记染色,同时设计维拉帕米对照组;（2）最佳分选喷嘴的确定：根据对数生长期的A549细胞处于不同的分裂时象的细胞大小,预实验确定最佳分选喷嘴;（3）光信号的调试：用RainbowBeads优化670/30[355]、460/50[355]荧光信号和FSC散射光信号,使光信号最强且变异系数（coefficient ofvariation,CV）最小.（4）最佳液滴延迟的确定：调节振荡频率（frequency）找出最佳液流断点位置,调节振幅（ampitude）优化侧液流信号直到两侧液流信号最稳定,Delay Calculator自动计算液滴延迟,根据Accudrop Beads的分选效果确定最佳液滴延迟;（5）其他指标的调整：调节液滴的充电电压（drop charge）和分选装置的X、Y轴坐标,使测试分选液滴准确达到分选装置中;设置分选参数,分选目的细胞群.结果：同一批次处理的A549肺腺癌细胞,100 μm的喷嘴为分选A549肺腺癌SP亚群的最佳喷嘴;CV值校正后SP比例为19.7％,提高了6％;液滴延迟校正后,其分选效率（efficiency）提高了15％,分选纯度（purity）提高了23％;10 μg/mL的Hoechst3342染色,其SP比例是5μg/mL染色的5倍以上;细胞浓度越稀,分选效率和分选纯度相应提高;活细胞比例越高,SP比例越高.结论：合适的喷嘴大小,是保证高分选效率和高分选纯度的基础;仪器较小的CV值和较精确的液滴延迟、合理的Hoechst33342染料浓度、较高的细胞存活率和适中的细胞浓度是保证高分选效率和高分选纯度的的关键;通过平衡上述影响分选的因素才可能得到最佳的分选效果.     

Design and application of a versatile triple-laser cell and chromosome sorter

A high-resolution triple-laser sorter was designed and constructed to provide flexible switchover and high-resolution sorting of cells or chromosomes with any combination of one, two, or three lasers. These features provide a central facility instrument that currently serves multiple users and analyzes different stain combinations with minimal switchover effort between experiments. Improved optics and mounts that focus the three laser beams independently are able to resolve beads and chromosomes better than our previously reported dual-laser sorter. An improved signal collection unit with electronically controlled reference positions can be focused more quickly and precisely for any signal combination. A removable dye laser extends the range of usable fluorochrome labels. A rapid sheath switchover permits sorting of sterile cells and sterile chromosomes sequentially without additional sterilization or reservoir sheath change. Improved dual-laser chromosome resolution is at least as good, analyzing 8,000 chromosomes/s, compared to the previous dual-laser bench at 2,000/s. Stimulated and unstimulated peripheral blood lymphocytes were analyzed according to simultaneous measurements of cell surface receptors labeled with a fluoresceinated neuropeptide and a Texas red-labeled antibody as well as DNA content during the cell cycle. These results demonstrate the broad range of potential applications of this triple-laser system.     

Induction of hypoxia in glass versus Permanox petri dishes

The survival of Chinese hamster ovary cells in culture following graded doses of X rays delivered under aerobic and hypoxic conditions, or treatment with the bioreductive drug SR 4233 under hypoxic conditions, was evaluated as a function of whether cells were plated onto glass or Permanox plastic petri dishes. In the case of treatment with SR 4233, the influence of varying the total volume of medium in the dishes was also studied. While the Permanox petri dishes were sufficient to yield "radiobiological" hypoxia, that is, oxygen enhancement ratios of approximately 3.0 were obtained for X irradiation, they were inferior to glass petri dishes with respect to the hypoxia-selective cytotoxicity of SR 4233. For a 90-min hypoxic exposure to 40 microM SR 4233, the surviving fraction of cells plated on plastic dishes averaged about 50-fold higher than that of cells plated on glass dishes. Although varying the total medium volume did affect the extent of SR 4233-induced cytotoxicity for glass dishes--drug toxicity decreased slightly with increasing medium volume--this was not the case for the plastic dishes, in which the cell survival following a fixed SR 4233 exposure was essentially constant as a function of medium volume. These results suggest, at least for SR 4233, and under these experimental conditions, that Permanox petri dishes are not satisfactory for such studies.     

High-speed photodamage cell selection using bromodeoxyuridine/Hoechst 33342 photosensitized cell killing

One of the major drawbacks of droplet sorting in a flow cytometer is the relatively low sorting speed. Thus, we have developed an alternative, faster sorting technique: photodamage cell sorting. In a photodamage cell sorter all unwanted cells, as detected with the first, measuring laser, are killed with the second, damaging laser. Thus, the cells need to be photosensitive to the second laser. In addition, a mechanism is needed to switch this laser on and off based on the sorting criteria. In our photodamage cell sorter, the ZAPPER, we use an acousto-optic crystal to switch the laser beam. Cells are made photosensitive by vital staining with photosensitizers. With cells grown in the presence of 5-bromo-2'-deoxyuridine (BrdUrd) and stained with Hoechst 33342 (H42) at least a 5-decade cell reduction is accomplished after irradiation with 400 mW UV light. With this system, sorting rates have been achieved of 30,000 cells per second. Due to the selection based on photodynamic killing, this sorting technique is restricted to the selection of viable cells. Photodamage cell sorting seems well suited for isolating viable cells occurring in low percentages or for the sorting of large numbers of cells. Another application can be the sorting of large or fragile cells.     

Rapid assay for cell age response to radiation by electronic volume flow cell sorting

A new technique is described for measuring cell survival as a function of cell cycle position using flow cytometric cell sorting on the basis of electronic volume signals. The sorting of cells into different cell age compartments is demonstrated for three different cell lines commonly used in radiobiological research. Using flow cytometric DNA content analysis and [3H]thymidine autoradiography of the sorted cell populations, we demonstrate that the resolution of the age compartment separation is as good as or better than that reported for other cell synchronizing techniques. The variation in cell survival as a function of position in the cell cycle after a single dose of radiation as measured by volume cell sorting is similar to that determined by other cell synchrony techniques. This new method has several advantages, including: no treatment of the cells is required, thus, this method is noncytotoxic; no cell cycle progression is needed to obtain different cell age compartments; the cell population can be held in complete growth medium at any desired temperature during sorting; and a complete radiation age-response assay can be plated in 2 h. The application of this method to problems in radiobiology and chemotherapy is discussed, along with some of the technical limitations.     

Electronic sorting of granulocyte precursor cells from stimulated bone marrow.

A commerical cell sorter was used to obtain preparations of cells in various stages of granulocyte development from rabbit marrows stimulated by inflammatory response. Marrow cells were fractionated on density gradients of Ficoll/Hypaque and each fraction sorted using light scatter. Trial and error selection of appropriate gradient fractions and light scatter windows allowed sorting of early (blast cells, promyelocytes), intermediate (myelocytes, metamyelocytes) and late stage (band cells, polys) granulocytes with enhanced purity.     

Sex preselection: high-speed flow cytometric sorting of X and Y sperm for maximum efficiency

Sex preselection that is based on flow-cytometric measurement of sperm DNA content to enable sorting of X- from Y-chromosome-bearing sperm has proven reproducible at various locations and with many species at greater than 90% purity. Offspring of the predetermined sex in both domestic animals and human beings have been born using this technology since its introduction in 1989. The method involves treating sperm with the fluorescent dye, Hoechst 33342, which binds to the DNA and then sorting them into X- and Y-bearing-sperm populations with a flow cytometer/cell sorter modified specifically for sperm. Sexed sperm are then used with differing semen delivery routes such as intra-uterine, intra-tubal, artificial insemination (deep-uterine and cervical), in vitro fertilization and embryo transfer, and intra-cytoplasmic sperm injection (ICSI). Offspring produced at all locations using the technology have been morphologically normal and reproductively capable in succeeding generations. With the advent of high-speed cell sorting technology and improved efficiency of sorting by a new sperm orienting nozzle, the efficiency of sexed sperm production is significantly enhanced. This paper describes development of the these technological improvements in the Beltsville Sexing Technology that has brought sexed sperm to a new level of application. Under typical conditions the high-speed sperm sorter with the orienting nozzle (HiSON) results in purities of 90% of X- and Y-bearing sperm at 6 million sperm per h for each population. Taken to its highest performance level, the HiSON has produced X-bearing-sperm populations at 85 to 90% purity in the production of up to 11 million X-bearing-sperm per h of sorting. In addition if one accepts a lower purity (75 to 80%) of X, nearly 20 million sperm can be sorted per h. The latter represents a 30 to 60-fold improvement over the 1989 sorting technology using rabbit sperm. It is anticipated that with instrument refinements the production capacity can be improved even further. The application of the current technology has led to much wider potential for practical usage through conventional and deep-uterine artificial insemination of many species, especially cattle. It also opens the possibility of utilizing sexed sperm for artificial insemination in swine once low-sperm-dose methods are perfected. Sexed sperm on demand has become a reality through the development of the HiSON system.     

Oxygen enhancement ratio as a function of dose and cell cycle phase for radiation-resistant and sensitive CHO cells

There is still controversy over whether the oxygen enhancement ratio (OER) varies as a function of dose and cell cycle phase. In the present study, the OER has been measured as a function of survival level and cell cycle phase using volume flow cell sorting. This method allows both the separation of cells in different stages of the cycle from an asynchronously growing population, and the precise plating of cells for accurate measurements at high survival levels. We have developed a cell suspension gassing and sampling system which maintained an oxygen tension less than 20 ppm throughout a series of sequential radiation doses. For both radiation-resistant cells (CHO-K1) and a radiation-sensitive clone (CHO-xrs6), we could separate relatively pure populations of G1-phase, G1/S-boundary, S-, and G2-phase cells. Each cell line showed a typical age response, with cells at the G1/S-phase boundary being 4 (CHO-K1) to 12 (CHO-xrs6) times more sensitive than cells in the late S phase. For both cell lines, G1-phase cells had an OER of 2.3-2.4, compared to an OER of 2.8-2.9 for S-phase and 2.6-2.7 for G2-phase cells. None of these age fractions showed a dependence of OER on survival level. Asynchronously growing cells or cells at the G1/S-phase boundary had an OER similar to that of G1-phase cells at high survival levels, but the OER increased with decreasing survival level to a value near that of S-phase cells. These results suggest that the decrease in OER at high survival levels for asynchronous cells may be due to differences in the OERs of the inherent cell age subpopulations. For cells in one cell cycle stage, oxygen appears to have a purely dose-modifying effect.     

Rapid isolation of single malaria parasite-infected red blood cells by cell sorting

Malaria research often requires isolation of individually infected red blood cells (RBCs) or of a homogenous parasite population derived from a single parasite (clone). Traditionally, isolation of individual, parasitized RBCs or parasite cloning is achieved by limiting dilution or micromanipulation. This protocol describes a method for more efficient cloning of the malaria parasite; the method uses a cell sorter to rapidly isolate Plasmodium falciparum-infected RBCs singly. By gating the parameters of forward-angle light scatter and side-angle light scatter in a cell sorter, singly infected RBCs can be isolated and automatically deposited into a 96-well culture plate within 1 min. Including a Percoll purification step; the entire procedure to seed a 96-well plate with singly infected RBCs can take <40 min. This highly efficient single-cell sorting protocol should be useful for cloning of both laboratory parasite populations from genetic manipulation experiments and clinical samples.     

Synchronization of murine erythroleukemic cells. Nuclear volume measurements for monitoring cell cycle traverse

Murine erythroleukemic cells (T3C12 clone) were synchronized either by using two 8-h thymidine (2 mM) blocks separated by a 7-h release period, or by centrifugal elutriation. To monitor the synchrony induction and cell cycle traverse, the cells were lysed with NP40 for 20 min and the resulting nuclei fixed with glutaraldehyde. These nuclear volume measurements were performed with a Coulter Electronics H4 cell volume spectrometer and compared with flow cytometric determinations of nuclear DNA content on a Coulter Electronics TPS-1 cell sorter. The results indicate that the nuclear volume measurements can be used as a simple precise method to monitor the cell cycle position and traverse of synchronized erythroleukemic cells.     

A UV‐C LED‐based unit for continuous decontamination of the sheath fluid in a flow‐cytometric cell sorter

Aseptic cell sorting is challenging, especially when a flow‐cytometric cell sorter is not operated in a sterile environment. The sheath fluid system of a cell sorter may be contaminated with germs such as bacteria, yeasts, viruses, or fungi. Thus, a regular chemical cleaning procedure is required to prepare a sorter for aseptic cell sorting by flushing the fluidic system. However, this procedure is time consuming, and most importantly, the researcher can never be sure that the cleaning process was successful. Here we present a method in which the sheath fluid of a cell sorter was decontaminated by irradiation with UV‐C light using a flow‐through principle. Using this principle, we were able to achieve a 5 log reduction of bacteria in the sheath fluid.     

Preservation of cells sorted individually onto microscope slides with a fluorescence-activated cell sorter

Fluorescence-activated cell sorters permit analyses and separation of cell populations based on light scatter and surface immunofluorescence parameters. Since a sorter can deposit individually identifiable cells onto a microscope slide, it was considered of interest to combine the flow measurements with analyses available on cells adhering to a surface as in, for example, morphological studies, cytoplasmic immunofluorescent staining, and mRNA in situ hybridization. A necessary condition for these studies is the preservation of cell structures after sorting. We report here a procedure suitable for this purpose. The most important features of this procedure are A) reducing the saline content of the sorter sheath fluid to about 0.0015 M (one-hundredth that of normal saline) to prevent cell damage due to hypertonicity during drying, and B) coating the substrate with a thin layer of newborn calf serum to promote the adherence of the cells to the substrate during subsequent fixing and staining.     

Multiparameter analysis and sorting of mammalian cells

An improved multisensor cell sorting instrument for quantitative analysis and sorting of cells has been developed. Cells stained with fluorescent dyes enter a flow chamber where cell volume, fluorescence, and light scatter sensors simultaneously measure multiple cellular properties. Cells then emerge in a liquid jet that is broken into uniform liquid droplets. Sensor signals are electronically processed in one of several ways for optimum cell discrimination and are displayed as pulse-amplitude distributions using a multichannel pulse-height analyzer. Processed signals activate cell sorting according to preselected parametric criteria by electrically charging droplets containing cells and electrostatically deflecting them into collection vessels. Illustrative examples of multiparameter cell analysis and sorting experiments using a model mouse tumor cell system, human and animal leukocytes, and cultured mammalian cells are presented.     

International Society for Analytical Cytology biosafety standard for sorting of unfixed cells.

BACKGROUND: Cell sorting of viable biological specimens has become very prevalent in laboratories involved in basic and clinical research. As these samples can contain infectious agents, precautions to protect instrument operators and the environment from hazards arising from the use of sorters are paramount. To this end the International Society of Analytical Cytology (ISAC) took a lead in establishing biosafety guidelines for sorting of unfixed cells (Schmid et al., Cytometry 1997;28:99-117). During the time period these recommendations have been available, they have become recognized worldwide as the standard practices and safety precautions for laboratories performing viable cell sorting experiments. However, the field of cytometry has progressed since 1997, and the document requires an update. METHODS: Initially, suggestions about the document format and content were discussed among members of the ISAC Biosafety Committee and were incorporated into a draft version that was sent to all committee members for review. Comments were collected, carefully considered, and incorporated as appropriate into a draft document that was posted on the ISAC web site to invite comments from the flow cytometry community at large. The revised document was then submitted to ISAC Council for review. Simultaneously, further comments were sought from newly-appointed ISAC Biosafety committee members. RESULTS: This safety standard for performing viable cell sorting experiments was recently generated. The document contains background information on the biohazard potential of sorting and the hazard classification of infectious agents as well as recommendations on (1) sample handling, (2) operator training and personal protection, (3) laboratory design, (4) cell sorter set-up, maintenance, and decontamination, and (5) testing the instrument for the efficiency of aerosol containment. CONCLUSIONS: This standard constitutes an updated and expanded revision of the 1997 biosafety guideline document. It is intended to provide laboratories involved in cell sorting with safety practices that take into account the enhanced hazard potential of high-speed sorting. Most importantly, it states that droplet-based sorting of infectious or hazardous biological material requires a higher level of containment than the one recommended for the risk group classification of the pathogen. The document also provides information on safety features of novel instrumentation, new options for personal protective equipment, and recently developed methods for testing the efficiency of aerosol containment.     

Improved methods for automatic monitoring of contracting heart cells in culture

The present communication describes improved methods for isolating and plating beating heart cells from neonatal rats using collagenase and collagen-coated petri dishes. The amplitude and frequency of contraction are continuously and simultaneously measured under well defined conditions and during prolonged periods of time with a highly sensitive and thermostated instrument. Additions, e.g. drugs and toxic agents, are made through an accessory pump system that involves extensive dilution of the added compound with medium; aliquots of medium can be withdrawn for estimation of metabolites. The system described is reliable and relatively inexpensive and allows a more extensive use of isolated heart cells, especially in studies of heart functions where small changes in amplitude and frequency of beating during prolonged periods of time are important.     

Factors to consider in performing survival studies with insect cells

Summary Insect cell lines are not well-suited to colony formation in liquid medium following low-density cell plating. The present studies demonstrate that the time of addition of fetal bovine serum to the culture medium and the number of γ-irradiated feeder cells added to each plate are important factors in developing a useful colony formation assay. TN-368 lepidopteran and WR69-DM-1 dipteran cell lines were used for these experiments. Both cell types display increased plating efficiencies if serum is added to the medium one or more days prior to plating as compared to adding serum immediately before plating. Growth curves obtained by seeding cells at higher densities also indicate that cell growth is slightly better if serum is added one or more days before seeding. These findings are especially important for survival and toxicity studies because the results demonstrate that even seemingly minor factors involved in cell survival assays may benefit treated cells to a greater degree than untreated control cells, thus providing an erroneous assessment of cell survival. This work was supported by USPHS grant R01-CA34158, awarded by the National Cancer Institute, DHHS, Bethesda, MD.     

Improving the biosafety of cell sorting by adaptation of a cell sorting system to a biosafety cabinet.

BACKGROUND: The jet-in-air cell sorters currently available are not very suitable for sorting potentially biohazardous material under optimal conditions because they do not protect operators and samples as recommended in the guidelines for safe biotechnology. To solve this problem we have adapted a cell sorting system to a special biosafety cabinet that satisfies the requirements for class II cabinets. With aid of this unit, sorting can be performed in conformance with the recommendations for biosafety level 2. METHODS: After integrating a modified fluorescence-activated cell sorter (FACS) Vantage into a special biosafety cabinet, we investigated the influence of the laminar air flow (LAF) inside the cabinet on side stream stability and the analytical precision of the cell sorter. In addition to the routine electronic counting of microparticles, we carried out tests on the containment of aerosols, using T4 bacteriophage as indicators, to demonstrate the efficiency of the biosafety cabinet for sorting experiments performed under biosafety level 2 conditions. RESULTS: The experiments showed that LAF, which is necessary to build up sterile conditions in a biosafety cabinet, does not influence the conditions for side stream stability or the analytical precision of the FACS Vantage cell sorting system. In addition, tests performed to assess aerosol containment during operation of the special biosafety cabinet demonstrated that the cabinet-integrated FACS Vantage unit (CIF) satisfies the conditions for class II cabinets. In the context of gene transfer experiments, the CIF facility was used to sort hematopoietic progenitor cells under biosafety level 2 conditions. CONCLUSIONS: The newly designed biosafety cabinet offers a practical modality for improving biosafety for operators and samples during cell sorting procedures. It can thus also be used for sorting experiments with genetically modified organisms in conformance with current biosafety regulations and guidelines.     

Flow cytometry and phytoplankton

Flow cytometry and sorting are now an important technology in aquatic research. Simultaneous measurements of individual particle cell size, fluorescence, and light scatter properties are directly applicable to current topics in aquatic research. Flow sorting may be employed to obtain subsets of cells for analysis by conventional methods. The manner in which rapid, precise measurements of single cells are made is complex, and the application of this technology to aquatic samples is subject to many analytical constraints. Flow cytometric measurements of algal cell size and pigment autofluorescence are relative and are therefore dependent on the optical configuration and variability of the instrument. Specific types of reference materials are used to establish the validity of analyses: 1) instrument standards, 2) fluorescence controls, and 3) internal stain standards. The selection and application of standards and controls are discussed in the context of allometric (cell size versus pigment fluorescence) and ataxonomic (pigment color groups) methods. The widespread acceptance of particular reference materials among research groups will result in comparable data sets describing aquatic particle distributions.