流式细胞光度术DNA分析在非何杰金氏淋巴瘤预后中的意义

用流式细胞光度术对一组非何杰金氏淋巴瘤(NHL)存档石蜡标本进行DNA分析。DNA异倍体肿瘤占总例数的47.4％,高、中度恶性组异倍体肿瘤的比例(76.8％和51.9％)分别高于低度恶性组(17.6％)(P<0.05)。S期细胞比例(synthetic phase fraction,SPF)随组织学恶性程度升高而增大,低、中和高度恶性淋巴瘤平均SPF值分别是6.5(0.6—18.8)％,13.5(3.2—37.3)％和23.4(4.4—41.4)％。二倍体和异倍体肿瘤患者的生存率无差别。全部病例中高SPF值肿瘤患者具有生存短的趋向(P=0.1)。低度恶性组内也存在此种趋向(P=0.08)。上述结果表明流式细胞光度术DNA分析对估价非何杰金氏淋巴瘤生物学行为是一种不依赖形态学的有用方法。     

大蒜油抗肿瘤作用的流式细胞光度术分析

用流式细胞光度术（ｆｌｏｗｅｙｔｏｍｅｔｒｙ,ＦＣＭ）+Ｓ０．２ｍｌ／日。吐温对照组ｌＰ吐温８００．２ｍｌ／日,以排除大蒜油制剂中溶剂（吐温８０）的影响。实验组ｌＰＧＯ１００ｍｇ／ｋｇ／．日。于连续４次注射后的６ｈ,２、３、５、１０天及１０次注射ＧＯ后７天取材,行ＦＣＭ样品制备。三、ＦＣＭ样品制备与测定中国中医研究院基础所腹水癌细胞用ＰＢＳ洗涤,７０％酒精固定,ＲＮａｓｅ消化细胞中的ＲＮＡ,ＰＩ染色。用腹腔内淋巴细胞作标准二倍体细胞。用４２０型荧光激活细胞分选仪（美国Ｂｅｃｔｏ－Ｄｉｃｋｓｏｎ公司产品）测单个肿瘤细胞的ＤＮＡ含量,用组方图显示肿瘤细胞ＤＮＡ倍体性质。图中第一个是ＤＮＡ二倍体（ＺＣ）峰,处于该峰的细胞为Ｇ;／Ｇ。期细胞,第二个峰为４ＣＤＮＡ峰,该处细胞为Ｇ。－Ｉ－－Ｍ期细胞,从２。到４。之间的细胞为Ｓ期细胞。大于４。ＤＮＡ峰细胞为高倍体细胞‘’：。根据不同峰值大小判定用药后肿瘤细胞倍体性质的变化。结果ＮＳ与吐温对照组肿瘤细胞都为非整倍体性,且呈多倍体性（图１,２）。连续４次给药后６ｈ,绝大部分多倍体肿瘤细胞被杀伤,残存肿瘤细胞以二倍体为主,多倍体肿瘤细胞峰值显著下降,甚至在组方图上几乎显不出多倍     

应用流式细胞仪定量评估人—鼠杂交瘤株细胞抗体生成功能稳定性的研究

用FACS分析和Luria-Delbrǔck方程计算淋巴细胞杂交瘤株细胞丧失抗体生成功能的突变机率(μ),以评估杂交瘤的稳定性。结果表明μ值大,则杂交瘤不稳定,易丧失抗体生成功能,反之则杂交瘤比较稳定。实验证明所建立的方法能反映杂交瘤稳定性的程度,具有实验少人为主观因素干扰,统计意义强,适用范围广等优点。     

CD11b/DNA双参数流式细胞术检测HL-60细胞分化的细胞周期

目的采用双参数流式细胞术研究全反式维甲酸(alltransretinoidacid,ATRA)诱导人类急性早幼粒白血病细胞HL-60细胞分化的细胞周期。方法HL-60细胞经分化诱导剂ATRA(终浓度为1μmol/L)诱导不同时间点后,利用CD11b/DNA双参数流式细胞术同时检测分化细胞表面抗原CD11b的表达及分化细胞DNA含量。结果HL-60细胞经ATRA诱导后,细胞表面分化抗原CD11b表达明显升高,细胞阻滞于G0/G1期,且CD11b阳性细胞主要位于G0/G1期。结论CD11b/DNA双参数流式细胞术能简便,快速,直观地检测细胞分化的细胞周期。     

IDENTIFICATION OF THE CLASS PRYMNESIOPHYCEAE AND THE GENUS PHAEOCYSTIS WITH RIBOSOMAL RNA–TARGETED NUCLEIC ACID PROBES DETECTED BY FLOW CYTOMETRY1

Target regions specific for the class Prymnesiophyceae and the genus Phaeocystis (Har.) Lag. were identified from 18S ribosomal RNA coding regions, and two complementary probes were designed (PRYMN01 and PHAEO01). Detection of whole cells hybridized with these probes labeled with fluorescein isothiocyanate was difficult using epifluorescence microscopy because autofluorescence of the chlorophylls seriously interfered with the fluorescence of the probes. In contrast, flow cytometry proved very useful to detect and quantify the fluorescence of the hybridized cells. Hybridization conditions were optimized, especially with respect to formamide concentration. Both probes were tested on a large array of both target and nontarget strains. Positive and negative controls were also analyzed. Specificity was tested by adding a competing nonlabeled probe. Whereas probe PHAEO01 seems to have good specificity, probe PRYMN01 appeared less specific and must be used with stringent positive and negative controls.     

FLOW CYTOMETRY AND MICROSCOPY OF GAMETOGENESIS IN NITZSCHIA PUNGENS,A TOXIC,BLOOM-FORMING,MARINE DIATOM1

The domoic acid-producing diatom Nitzschia pungens Grunow f. multiseries Hasle, which is responsible for amnesic shellfish poisonings in Prince Edward Island, Canada, underwent gametogenesis when senescent cells (i.e. in stationary growth phase for more than 290 days) were subcultured into fresh FE medium and light intensity was increased from 33 to 530 μE · m^?2· s^?1. The number of gametes produced varied with the salinity of the medium, with a maximum at 23.5‰. Cells in the exponential growth phase (0.8 div · d^?1) did not produce gametes, nor did senescent cells when transferred without change in light intensity. Anisogamous gametes, probably haploid, were isolated by combining conventional microscopy with flow cytometry. Zygotes resulting from syngamy yielded cigar-shaped naviculoid cells, morphologically different from parent cells (heteromorphism). These cells, with a division rate of 1.9 div · d^?1, could serve as a seed population and explain the origin and rapid progression of the toxic blooms of red-water proportions that have been a public health problem in Eastern Canada. Production of domoic acid by postexponential and moribund cells but not by gametes, zygotes, or immediately resulting cells, provides an insight into the dependence of toxicity on the developmental history of this diatom.     

CELL AND GROWTH CHARACTERISTICS OF TYPES A AND B OF EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) AS DETERMINED BY FLOW CYTOMETRY AND CHEMICAL ANALYSES1

Two morphotypes of Emiliania huxleyi (Lohmann 1902) Hay et al. 1967, types A and B, known to be unequally distributed in the oceans, were grown in dilution cultures at a range of photon flux densities (PFDs) (1.5–155 μmol photons·m^?2·s^?1) and two temperatures (10° and 15° C). Calcite carbon and organic carbon content of the cells as well as instantaneous growth rate, cell size, chlorophyll fluorescence, and light-scatter properties clearly depended on growth conditions and differed considerably for the two morphotypes. The ratio between calcite carbon and organic carbon production showed an optimum of 0.65 in E. huxleyi type A cells at PFD = 17.5. The ratio increased slightly with a temperature increase from 10° to 15°C but remained < 1.0 at both temperatures in light-limited cells. In contrast, calcite carbon production exceeded organic carbon production (ratio: 1.4–2.2) in phosphate-deprived cultures. Emiliania huxleyi type B generally showed a higher calcite carbon/organic carbon ratio than E. huxleyi type A, but the relation with PFD was similar. The content of calcite carbon and organic carbon as well as the instantaneous growth rate, cell size, chlorophyll fluorescence, and light-scatter properties showed large diel variations that were closely related to the division cycle. Our results show the importance of mapping the structure of any sampled cell population with respect to the phase in the cell division cycle, as this largely determines the outcome of not only “per cell” measurements but also short time (less than 24 h) flux measurements. For instance, dark production of calcite by E. huxleyi was negatively affected by cell division. Slowly growing (phosphate-stressed) cultures produced calcite in the light and in the dark. In contrast, rapidly growing cultures at 10°C produced calcite only in the light, whereas in the dark there was a significant loss of calcite due to dissolution.     

小儿急性白血病细胞DNA、蛋白质含量及其临床意义

用流式细胞术（ＦＣＭ）测定了４５例不同病期急性白血病患儿和１３例非白血病患儿骨髓细胞ＤＮＡ及蛋白质含量,结果显示：１．急性白血病患儿的ＤＮＡ非整倍体检出率为４１．２％,其中ＡＬＬ为３３．３％,ＡＮＬＬ为６０％;２．ＡＬＬ组和ＡＮＬＬ组的ＤＮＡ合成期细胞百分数（Ｓ％）显著低于非白血病组,而ＣＲ组与非白血病组之间的Ｓ％差异无显著性;３．ＡＮＬＬ组的ＰｒＩ显著高于ＡＬＬ组、非白血病组及ＣＲ组,ＡＬＬ的ＰｒＩ显著低于ＣＲ组和非白血病组,而非白血病组与ＣＲ组的ＰｒＩ差异无显著性。四组之间ＬＰＣ－Ｆ差异无显著性;４．在对１１例ＣＲ病人进行的连续观察过程中,５例病人检出ＤＮＡ非整信体,其中４例于检出ＤＮＡ非整倍体后３３～７４天复发。     

流式双参数检测在肿瘤增殖,分化与凋亡中的应用

流式细胞术已在生物医学研究中有广泛应用,双参数流式术显著特点是给同一细胞群提供双重信息。本文成功地将这一先进技术用于食管癌增殖、分化与凋亡的检测,显示流式双参数的广阔应用前景。     

DETERMINATION OF NON-PROLIFERATING CELLS IN CULTURE BY COMBINING FLOW CYTOMETRY WITH STATHMOKINETICS

Colcemid was added to the growth medium of L-cells in monolayer culture. The proliferating cells continued their progression through the cycle up to metaphase, where they were arrested. At different times after Colcemid addition the cells were trypsinized. suspended immediately in a solution of the DNA-specific fluorescent dye Hoechst 33258 and analysed with a flow cytometer. The histograms were evaluated to give the fraction of cells in the 2c peak as a function of time after Colcemid addition. The flux into the 2c compartment being interrupted, the peak content decreased until all proliferating (G₁) cells had entered S-phase. With increasing cell density or with increasing time after serum deprivation an increasing fraction of cells remained in the 2c peak at times greater than the normal G₁ duration. The possibility of applying this method to the determination of non-proliferating cells in a population is discussed.     

SUGAR-CONTAINING COMPOUNDS ON THE CELL SURFACES OF MARINE DIATOMS MEASURED USING CONCANAVALIN A AND FLOW CYTOMETRY

Some diatom exudates may remain attached to the exterior cell surface, potentially altering cell stickiness and affecting important aspects of the diatom's ecology such as aggregation rates and grazing rates. We measured the accumulation of cell-surface sugar-containing compounds by labeling cultured marine diatoms with fluorescent-tagged sugar-binding lectins and measuring the fluorescence associated with each cell using flow cytometry. The binding of FITC-labeled concanavalin A (FITC-ConA), a lectin that binds to glucose and mannose residues, varied 5-fold among different diatom species in exponential growth (on a per-cell basis) and 2–3-fold within a given species in different physiological states. Although transparent exopolymer particles followed a simple accumulation curve over time in batch culture, FITC-ConA. cell^-1 did not follow the same pattern, suggesting that surface sugar accumulation is not driven simply by the accumulation of such particles in the medium. For Thalassiosira pseudonana (Hustedt) Hasle and Heimdal (3H clone), the amount of sugar-containing compounds on the cell surface increased transiently as growth rate slowed in early stationary phase under both N and Si limitation. For Chaetoceros neogracile (Schuett) van Landingham, FITC-ConA. cell^-1had a strong inverse relationship with growth rate across several Si-limited batch culture experiments. Both results suggest some biological mediation of cell-surface sugar-containing compounds. Our study reveals the great potential for using lectin binding to investigate cell-surface sugars on diatoms. Lectins allow us to investigate noninvasively the role of cell-surface sugar-containing compounds in modifying cell stickiness and aggregation, as well as the partitioning of exuded phytoplankton carbon. We suggest that cell-surface sugar accumulation may be related to diatom stickiness, based on a correlation between our FITC-ConA measurements and stickiness estimates in the literature on several of the species we studied.     

CELL-PRODUCTION RATES ESTIMATED BY THE USE OF VINCRISTINE SULPHATE AND FLOW CYTOMETRY

A new method for the evaluation of cell production rates combining flow cytometry (FCM) and the stathmokinetic method using vineristine sulphate (VS) has been used for the analysis of three aneuploid ascites tumours at different stages of growth. Using this technique it was possible to estimate the well-known decrease in cell production rates of ageing ascites tumours. The percentage of normal host cells in the aneuploid tumours studied was easily determined by FCM prior to the calculation of the tumour cell-production rates. A correlation was found between the percentage of tumour cells in the S phase and the tumour cell-production rate. This correlation is probably explained by the gradual transfer of proliferating cells in S phase to resting G₁ and G₂ phases with increasing tumour age.     

COMPARISON OF PLANT DNA CONTENTS DETERMINED BY FEULGEN MICROSPECTROPHOTOMETRY AND LASER FLOW CYTOMETRY

An improved procedure is reported for determining DNA amounts of plant nuclei. Nuclei stained with propidium iodide, isolated from chopped plant leaves, were passed through an Ortho Cytofluorograph with a Lexel model 95 argon laser (514 nm) and the fluorescence measured, integrated, and recorded using an Ortho 2140 Data Acquisition computer. All nuclear samples were mixed with nuclei of Sultan barley (2C DNA content = 11.12 pg [picogram]) as an internal standard. DNA contents of ten plant species, ranging from 2C = 1.7 pg to 36.1 pg measured by flow cytometry, correlated strongly (r = 0.99, slope = + 1.00) with DNA contents determined from Feulgen-stained nuclei of the same species using microspectrophotometry. The flow cytometric procedures were sufficiently sensitive to detect differences in DNA content between inbred lines of corn and their F1 hybrids. Our results obtained with improved procedures, specifically using propidium iodide as a fluorochrome and plant nuclei instead of chicken erythrocytes as an internal standard, demonstrate that laser flow cytometry can be a precise, rapid, and reliable method for determining nuclear DNA content of plants.     

FLOW CYTOMETRIC ANALYSIS OF SPERMATOGENESIS IN THE DIATOM THALASSIOSIRA WEISSFLOGII (BACILLARIOPHYCEAE)1

Flow cytometry was used to detect and quantify sexual differentiation in the centric diatom Thalassiosira weissflogii (Grun.). Size (light scatter), chlorophyll, protein and DNA contents were measured for each cell throughout the process of differentiation. Male gametes were small round cells characterized by one complement of DNA and a lower protein and chlorophyll content than vegetative cells. Male gamete formation was induced by a long period of darkness (2 days) followed by a transfer to continuous light. Up to 30% of the initial cell population produced male gametes which appeared in the culture 14 h after release from darkness. Male gamete production was also detected in exponentially growing cultures in continuous light, but to a much smaller degree.     

FLOW CYTOMETRIC ANALYSIS OF SPERMATOGENESIS IN THE DIATOM THALASSIOSIRA WEISSFLOGII (BACILLARIOPHYCEAE)1

Flow cytometry was used to detect and quantify sexual differentiation in the centric diatom Thalassiosira weissflogii (Grun.). Size (light scatter), chlorophyll, protein and DNA contents were measured for each cell throughout the process of differentiation. Male gametes were small round cells characterized by one complement of DNA and a lower protein and chlorophyll content than vegetative cells. Male gamete formation was induced by a long period of darkness (2 days) followed by a transfer to continuous light. Up to 30% of the initial cell population produced male gametes which appeared in the culture 14 h after release from darkness. Male gamete production was also detected in exponentially growing cultures in continuous light, but to a much smaller degree.     

IN-SITU MORPHOLOGY AND OCCURRENCE OF EUCARYOTIC PHOTOTROPHS OF BACTERIAL SIZE IN THE PICOPLANKTON OF ESTUARINE AND OCEANIC WATERS1

Concentrates of the picoplankton (0.2–2.0 μm) sized fraction from the euphotic zone of estuarine and oceanic waters were examined by transmission electron microscopy. In addition to the numerous phototrophic procaryotes (chroococcoid cyanobacteria) previously reported, small phototrophic eucaryotes were observed in 20 of 25 samples examined. Micromonas pusilla (Butcher) Manton and Parks, a 1 × 1.5 μm flagellate, was abundant in estuarine samples in summer. Similar sized cells of non-flagellated chlorophytes, either Nannochloris Naumann or Chlorella Beijerinck, were observed sporadically in many samples. The most ubiquitous microalga was a scaled, non-flagellated prasinophyte that occurred at 9 of 15 different locations on 15 of 20 sampling dates in water samples from Iceland to the Caribbean Sea, This tiny alga (0.5 to 1.0 μm in diam.) is probably the smallest known photo-trophic eucaryote and has not heretofore been described. Enrichment cultures using conventional techniques on several cruises yielded only the Chlorella-type of green alga, as well as numerous isolates of unicellular chroococcoid cyanobacteria.     

ACTIVE GROWTH OF THE OCEANIC DINOFLAGELLATE CERATIUM TERES IN THE CARIBBEAN AND SARGASSO SEAS ESTIMATED BY CELL CYCLE ANALYSIS1

The growth rate of an oceanic dinoflagellate, Ceratium teres Kofoid, was investigated in the Sargasso and Caribbean Seas from September 1989 to July 1990 using the cell cycle analysis method. Estimated growth rates ranged from 0.29 to 0.58 day^?1 and were 1.5–7.2 times higher than generally accepted rates for oceanic dinoflagellates. The higher rates in this report were mainly due to an improvement in techniques that determine the duration of a terminal cell cycle phase in situ. The day-to-day variation in growth rates was surprisingly small, but, from long-term measurements, a weak correlation was found among temperature, daily irradiance, and seasonal growth rate. The calculated species-specific primary production ranged from 0.5 to 1.8 mg C·m^?2·day^?1, about 1% of the estimated total production. Ceratium teres may be an important carbon source at the base of the grazing food chain.     

ISOLATION AND FLOW CYTOMETRIC CHARACTERIZATION OF PROTOPLASTS FROM MARINE MACROALGAE

Protoplasts were isolated from Ulva rigida C. Agardh (Chlorophyta) and two species of Rhodophyta , Gracilariopsis lemaneiformis ( Bory) Dawson, Acleto et Folvik and Gracilaria tenuistipitata Chang et Xia var . liui with minor modifications (the inclusion of 0.01% agarase in the set of cell-wall-degrading enzymes for the two red algae). Flow cytometric characteristics of freshly isolated protoplasts were determined on a FACScan flow cytometer (FC). The most useful parameters for characterizing protoplasts from marine algae were forward angle light scatter (FSC), orange fluorescence (FL2) and red fluorescence (FL3). Protoplasts from all the species were easily distinguishable when their FSC, FL2, and FL3 signals were combined in the bivariate plots FL3 vs. FSC and FL3 us. FL2. Two alternative techniques to help identify protoplasts from debris in the FC computer screen were developed (for FC without sorting capability). Both techniques were based on the ability of new FCs to record time. The first one was based on the induction of rapid changes of cell volume in response to osmotic stress. Only intact protoplasts responded to changes in the osmotic pressure. The second one was based on the uptake and hydrolysis of fluorescein diacetate by intracellular esterases. Viable protoplasts showed a hyperbolic accumulation of fluorescein with time. Semimaximal fluorescein accumulation was attained in 30.5 ± 9.5 s. Debris was easily recognized since, contrary to protoplasts, it did not show a time-dependent accumulation of fluorescein .