SCI论文写作典型句型（2）——细胞培养篇

SCI 论文写作典型句型 2 细胞培养篇 点击上面蓝字 关注 解螺旋 获得标本获得标本 Twenty two postmortem eyes from thirteen donors aged 30 67 yr mean SD 57 11 yr were obtained from somewhere city country Iridectomy specimens were obtained from eyes undergoing glaucomafiltration surgery at the hospital Of the 65 specimens 12 were obtained frompatients with A age 57 88 years and 53 were obtained from patients without A age 3 months to 82 years 知情同意书知情同意书 All tissues were obtained with pre mortem consent in accordance with thelaws and regulations in place in the various jurisdictions Institutional guidelines regarding animal experimentation were followed 细胞来源细胞来源 The cells used in this study consisted of four cell lines two isolatedfrom the iris and two from the choroid 细胞培养 细胞培养 分离组织分离组织 A were isolated andcultured from adult donor eyes as described previously Briefly acircumferential scleral incision was made in the sclera 8 mm behind the limbus atthe ora serrata separating the globe into anterior and posterior portions The anterior portionof the globe The posterior segment including were excised and placed in aculture dish The iris was excisedat the root placed in a culture dish with its posterior inner surfaceupward facing upward washed with and covered with immersed in for 1hr at 37 C The ciliary epithelium wasseparated from the ciliary body after immersion with trypsin solution for 2 to3 hours was then separated dissect from under the a dissecting microscope a stereo microscope The iris was cut into2 or 3 small pieces that were plated on to a 35 mm Falcon dish with 0 5 mL ofmedium The conjunctiva andunderlying sclera were dissected from the donor eyes or the rims put in aculture dish immersed with 0 2 dispase solution and cultured at 37 C for 1 5h 酶消化酶消化 solution was addedand the posterior segment incubated for 1 hr at 37 C The RPE was thenseparated from under the a dissecting microscope after immersion in 0 25 trypsin solution at 37 C for 1 to 2 hours The uveal stroma was treatedby trypsin as just described 18 hr at 4 C and 1 hr at 37 C The trypsin solution wasreplaced by collagenase solution 400 U ml in F 12 medium Sigma St Louis MO and incubated at 37 C The iris was immersedin 1 of 2 enzyme solutions 0 25 trypsin Gibco or 0 12 trypsin supplementedwith 2 5 mg mL pancreatin Gibco and 200 U mL collagenase Sigma St Louis Mo TCP solution The iris was incubatedat 37 C for 2 to 4 hours until the IPE detached from the stroma 细胞收集细胞收集 The isolated cells werecollected with a pipette The cells releasedwere collected centrifuged resuspended with culture medium and plated intoFalcon culture flasks 中和 Trypsin activity wasstopped by adding culture medium with 10 fetal bovine serum The Ham F12 medium which was supplemented with 10 fetal bovine serum was added to the IPE cellsuspensions isolated by methods 离心离心 接种接种 The cells released Theisolated cells were collected centrifugated resuspended with culture medium and plated in Falcon culture dishes The collagenasesolution was replaced and released cells were collected centrifuged resuspended and plated each hour for 3 hours The isolated uvealsegments were placed in centrifuge tubes and UM were isolated by method Each type of cell wasplated into 18 wells of 24 well culture plates The suspensions werecentrifuged resuspended in a culture medium plated onto Falcon culturedishes and incubated in a humidified 95 air 5 CO2 atmosphere Cells isolated bythese two different methods were plated to two wells 多种方法比较多种方法比较 The number and the growth capacity of cells obtained from these twodifferent methods were measured for comparison The number of isolated and attached cells for these two groups werecalculated and expressed as number per eye separately for comparison 培养培养 The isolated Awerecultured in Falcon culture dishes with FIC medium which consisted of F 12medium supplemented with 10 fetal bovine serum 2 mM glutamine all fromGibco 10 ng ml cholera toxin 50 mg ml gentamicin all from Sigma and 20 ng ml basic fibroblast growth factor The isolated cells were incubated in a CO2 regulated incubator inhumidified 95 air 5 CO2 atmosphere One of these three culture media wasused 1 F12 medium Ham s F12 Nutrient Mixture supplemented by 10 FBS gentamicin 50 mg ml and glutamine 2 mM Gibco 2 TIC medium F12 medium was supplemented by 100 nM TPA 0 1 mM and10 ng ml CT Sigma 3 FIC medium TIC medium was prepared as above except that 20 ng ml humanrecombinant basic fibroblast growth factor was substituted for TPA Additional culture medium was added 1 to 2 hours later 0 5 mL and again24 hours later 1 mL 观察观察 Cultures were observed daily with a inverted phase contrast microscope 换液 传代换液 传代 The medium was changedthree times a week Culture media werereplaced two times per week every 2 days After 24 hr the medium was replaced with 0 5 ml of test media Test mediaconsisted of 1 After primary cultures became confluent the cells were detached by usinga 0 125 trypsin 0 01 edetic acid solution counted centrifugated diluted1 2 to 1 4 and plated for subculture The same culture medium was used for the subcultures except that theconcentration of fetal bovine serum was changed from 30 to 20 Cell lineswere passaged at intervals of 3 to 7 days routinely at a dilution of 1 2 to1 3 every 5 to 10 days The isolated cells used had been in culture for less than 2 months and hadbeen passaged three to six times at a dilution of 1 3 1 4 Early passages of cultured Cs were plated into 24 well plates with FICmedium at a density of 1 2 104 per well The cells were then detached by trypsin and counted using a hemocytometer 细胞污染细胞污染 In 11 specimens the IPE grew from some explants but was frequentlycontaminated with other cell types No pure confluent primary culture wasobtained by this method 清除污染细胞清除污染细胞 To eliminate contaminating cells when necessary geneticin was added tothe culture medium to a final concentration of 100 mg ml The cultureswere subjected to this treatment for 3 7 d replacing the medium twice a week Geneticin a cytotoxicagent was added 100 mg ml for 3 to 7 days whennecessary to eliminate contaminating cells 生长速度生长速度 Growth rate The growth rate is the percentage of cells entering thedividing phase in the total population of plated cells ratio of dividing cellsto plated cells and it was calculated from the difference in the number ofplated cells and stationary cells In 4 cultures passaged continuously until senescence the mean cumulativepopulation doubling was and the mean doubling time was days Doubling time and population doublings are calculated as describedpreviously The mean cell number in confluent primary cultures was and thepopulation doubling of growing IPE cells in primary cultures was 7 78 Plating efficiency attached cells plated cells Nonattached cells in thefluid withdrawn from the culture during the first and second replacements ofmedium were counted The difference between the number of plated cells anddetached cells is the number of attached cells Spreading rate spread cells plated cells After spreading the nucleiand the cytoplasm of the UM could be recognized separately The spread andnonspread cells in 5 10 microscopic fields were counted to estimate theproportion of spread cells Percentage of stationary cells Stationary cells were heavily pigmented whereas dividing cells had minimal pigment Comparing of the number ofpigmented cells at t