一种制备新生隐球菌单细胞的方法
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摘要
【目的】建立流式细胞仪分选新生隐球菌单细胞的方法,确定新生隐球菌单细胞恢复生长的条件和能力。【方法】利用Moflo XDP流式细胞分析分选仪,体外测定不同条件下新生隐球菌恢复生长的比率。【结果】建立了流式细胞仪新生隐球菌单细胞分选流程。确定流式细胞仪分选得到的新生隐球菌单细胞具有恢复生长的能力,恢复生长的能力受营养条件和菌株差异的影响。在营养丰富的条件下,新生隐球菌JEC21和H99单细胞恢复生长比率分别为74%和89%。在寡营养条件下,JEC21和H99单细胞恢复生长比率分别为37%和80%。JEC21生长比率随细胞数的增加而升高,细胞数为100个时,生长比率为55%;细胞数为1000个时,生长比率为97%。【结论】流式细胞仪分选得到新生隐球菌单细胞具有恢复生长的能力,生长能力受营养条件、菌株差异的影响。
[Objective] A method of sorting single cells of Cryptococcus neoformans by flow cytometry was established to determine factors influencing proliferation of single C. neoformans cells. [Methods] The Moflo XDP flow cytometric sorter was used. The clonal recovery rate of C. neoformans cell was determined under various conditions. [Results] Single C. neoformans cells sorting process by cytometry was established. Single C. neoformans cells could recover growth which was affected by nutritional conditions and strains. Under nutrient-rich conditions, the clonal recovery rates of C. neoformans stains JEC21 and H99 were 74% and 88%, respectively. Under oligotrophic conditions, the clonal recovery rate of JEC21 was 37%; while the clonal recovery rate of H99 was 80%. The clonal recovery rate of JEC21 increased with increasing initial number of cells. When the cell number of JEC21 strain was 100, the clonal recovery rate was 55%; when the cell number was 1000, the clonal recovery rate was 97%. [Conclusion] The single C. neofroman cells sorted by flow cytometry had the capacity of recovery to growth, and the capacity was affected by nutritional conditions and strains.
[Objective] A method of sorting single cells of Cryptococcus neoformans by flow cytometry was established to determine factors influencing proliferation of single C. neoformans cells. [Methods] The Moflo XDP flow cytometric sorter was used. The clonal recovery rate of C. neoformans cell was determined under various conditions. [Results] Single C. neoformans cells sorting process by cytometry was established. Single C. neoformans cells could recover growth which was affected by nutritional conditions and strains. Under nutrient-rich conditions, the clonal recovery rates of C. neoformans stains JEC21 and H99 were 74% and 88%, respectively. Under oligotrophic conditions, the clonal recovery rate of JEC21 was 37%; while the clonal recovery rate of H99 was 80%. The clonal recovery rate of JEC21 increased with increasing initial number of cells. When the cell number of JEC21 strain was 100, the clonal recovery rate was 55%; when the cell number was 1000, the clonal recovery rate was 97%. [Conclusion] The single C. neofroman cells sorted by flow cytometry had the capacity of recovery to growth, and the capacity was affected by nutritional conditions and strains.
引文
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[2]Fisher MC,Henk DA,Briggs CJ,Brownstein JS,Madoff LC,McCraw SL,Gurr SJ.Emerging fungal threats to animal,plant and ecosystem health.Nature,2012,484(7393):186-194.
[3]Rajasingham R,Smith RM,Park BJ,Jarvis JN,Govender NP,Chiller TM,Denning DW,Loyse A,Boulware DR.Global burden of disease of HIV-associated cryptococcal meningitis:an updated analysis.The Lancet Infectious Diseases,2017,17(8):873-881.
[4]Jones KE,Patel NG,Levy MA,Storeygard A,Balk D,Gittleman JL,Daszak P.Global trends in emerging infectious diseases.Nature,2008,451(7181):990-993.
[5]Crespi BJ.The evolution of social behavior in microorganisms.Trends in Ecology&Evolution,2001,16(4):178-183.
[6]Lee H,Chang YC,Nardone G,Kwon-Chung KJ.TUP1disruption in Cryptococcus neoformans uncovers a peptide-mediated density-dependent growth phenomenon that mimics quorum sensing.Molecular Microbiology,2007,64(3):591-601.
[7]Wang LQ,Lin XR.The morphotype heterogeneity in Cryptococcus neoformans.Current Opinion in Microbiology,2015,26:60-64.
[8]Dark MJ.Whole-genome sequencing in bacteriology:state of the art.Infection and Drug Resistance,2013,6:115-123.
[9]Zhang L,Cui X,Schmitt K,Hubert R,Navidi W,Arnheim N.Whole genome amplification from a single cell:implications for genetic analysis.Proceedings of the National Academy of Sciences of the United States of America,1992,89(13):5847-5851.
[10]Fu YS,Li CM,Lu SJ,Zhou WX,Tang FC,Xie XS,Huang YY.Uniform and accurate single-cell sequencing based on emulsion whole-genome amplification.Proceedings of the National Academy of Sciences of the United States of America,2015,112(38):11923-11928.
[11]Kwon-Chung K.A new genus,Filobasidiella,the perfect state of Cryptococcus neoformans.Mycologia,1975,67(6):1197-1200.
[12]Qiao YX,Zhao XY,Zhu J,Tu R,Dong LB,Wang L,Dong ZY,Wang QH,Du WB.Fluorescence-activated droplet sorting of lipolytic microorganisms using a compact optical system.Lab on a Chip,2018,18(1):190-196.
[13]Lau AY,Lee LP,Chan JW.An integrated optofluidic platform for Raman-activated cell sorting.Lab on a Chip,2008,8(7):1116-1120.
[14]Spitzer MH,Nolan GP.Mass cytometry:single cells,many features.Cell,2016,165(4):780-791.
[15]Shapiro HM.Practical Flow Cytometry.4th ed.New York:John Wiley&Sons,2005.
[16]Vives-Rego J,Lebaron P,Nebe-von Caron G.Current and future applications of flow cytometry in aquatic microbiology.FEMS Microbiology Reviews,2000,24(4):429-448.
[17]Barer R,Saunders-Singer AE.A new single-control micromanipulator.Journal of Cell Science,1948,89(Pt 4):439-447.
[18]Nir R,Lamed R,Gueta L,Sahar E.Single-cell entrapment and microcolony development within uniform microspheres amenable to flow cytometry.Applied and Environmental Microbiology,1990,56(9):2870-2875.