用流式細(xì)胞儀分析脂肪細(xì)胞

1、analysis and isolation of adipocytes by flow cytometryyang li 2016-04-05majka sm, miller hl, helm km, et al. analysis and isolation of adipocytes by flow cytometry. methods in enzymology. 2014;537:281-296. doi:10.1016/b978-0-12-411619-1.00015-x.2backgroundaim of the researchdifficulty solutionwhat a

2、re adipocytes (fat cells)?major cells in the adipose tissue.3lipid droplettwo kinds of adipocytes in human4prospects of adipocyte biologylipid store inhibit eating increase metabolismleptin5prospects of adipocyte biologyadipokinesinvolved in energy metabolismlipid store 6prospects of adipocyte biolo

3、gyspecifically from adipose tissue lipid store adipokinesenergy metabolism7prospects of adipocyte biologyadipokines play an significant role in biological function.8flow cytometry (fcm)principle diagram9volume,morphological complexity of cells, total dna/rna content, apoptosis, cell viability, . . .

4、 .10a method for analysis and isolation free-floating adipocytes by flow cytometry.analysisflow cytometry (fcm)confocal laser scanning microscope (clsm)isolation fluorescence-activated cell sorting (facs)11analysisflow cytometry (fcm)confocal laser scanning microscope (clsm)beckman coulter-moflo xdp

5、 analysis rate:100,000 events/sec. sort rate: 70,000 events/sec.12a method for analysis and isolation free-floating adipocytes by flow cytometry.analysisflow cytometry (fcm)confocal laser scanning microscope (clsm)quantitivehigh resolution imaging 13a method for analysis and isolation free-floating

6、adipocytes by flow cytometry.analysisflow cytometry (fcm)confocal laser scanning microscope (clsm)imaging flow cytometer(amnis) 12 images for every cellquantitivehigh resolution imaging 14a method for analysis and isolation free-floating adipocytes by flow cytometry.isolation fluorescence-activated

7、cell sorting (facs)a passing cell can be selectively add different charge, falling into different containers.15a method for analysis and isolation free-floating adipocytes by flow cytometry.adipocytes are large and fragile 50200 m easy to be broken, when the flow rate (pressure) is large.16adipocyte

8、s are large and fragile 50200 m easy to be broken, when the flow rate (pressure) is large.contaminants stromal/vascular cells aggregates of adipocytes free lipid droplets other debris 17adipocytes are large and fragile 50200 m using flow cytometry with larger internal diameter of the fluidics (150 t

9、o 250m). easy to be brokencontrol flow pressures to 510 psi, slow but not broken. good for isolation.18contaminants stromal/vascular cells aggregates of adipocytes free lipid droplets other debris exclusion of contaminants (4 steps)19 collagenase digestion filtration undigested tissue fragments are

10、excluded. centrifugation separated from stromal/vascular cells fragile use rocking rather than vortexingpreparation of single cell suspension of free-floating adipocytes20step 1. initial separationby size adipocytes (50200 m) bigger than stromal/vascular cells(20 m) higher in forward scatter (fsc) t

11、he presence of highly refractile lipid droplets in adipocyte higher in ssc overlap need further separation small adipocytes ignored (entire analyze if necessary)21contaminants stromal/vascular cells aggregates of adipocytes free lipid droplets other debris exclusion of contaminants (4 steps)22step 2

12、. exclusion of aggregates and selection of singlets aggregates can be identified by dot plot (width or area). 3 parameters: height voltage, width passing time(size), area= h x w when aggregates(doublets) pass, spend more time, w increased 23step 2. exclusion of aggregates and selection of singlets s

13、tain nuclei by dyecycle violet (dcv) fluorescence. single cells can be easily gated from aggregates & other debris this step couldnt exclude remaining stromal/vascular cells.24step 3. exclusion of single stromal/vascular cells stain the lipid droplets by lipidtox deep red (lpdtx) positive select

14、ion of events containing lipid droplets25step 4: exclusion of remaining stromal contaminants a small probability event: some stromal cells may adhere to the free lipid droplets. the remaining stromal contaminants bearing the fluorescent antibodies (phycoerythrin(pe)-conjugated) to stromal lineage markers are stained to exclude.(negative selection)nega