用流式细胞仪分析脂肪细胞

1、精选课件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 Solution精选

2、课件What are adipocytes (fat cells)?Major cells in the adipose tissue.3精选课件Lipid dropletTwo kinds of adipocytes in human4精选课件Prospects of adipocyte biologyLipid store Inhibit eating Increase metabolismLeptin5精选课件Prospects of adipocyte biologyAdipokinesInvolved in energy metabolismLipid store 6精选课件Pros

3、pects of adipocyte biologySpecifically from adipose tissue Lipid store AdipokinesEnergy metabolism7精选课件Prospects of adipocyte biologyAdipokines play an significant role in biological function.8精选课件Flow Cytometry (FCM)Principle diagram9精选课件Volume,Morphological complexity of cells, Total DNA/RNA conte

4、nt, Apoptosis, Cell viability, . . . .10精选课件A 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)11精选课件AnalysisFlow cytometry (FCM)Confocal laser scannin

5、g microscope (CLSM)Beckman Coulter-MoFlo XDP Analysis rate:100,000 events/sec. Sort rate: 70,000 events/sec.12A method for analysis and isolation free-floating adipocytes by Flow Cytometry.精选课件AnalysisFlow cytometry (FCM)Confocal laser scanning microscope (CLSM)QuantitiveHigh resolution imaging 13A

6、method for analysis and isolation free-floating adipocytes by Flow Cytometry.精选课件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

7、 by Flow Cytometry.精选课件Isolation Fluorescence-activated 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.精选课件Adipocytes are large and fragile 50200 m Easy to b

8、e broken, when the flow rate (pressure) is large.16精选课件Adipocytes 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 17精选课件Adipocytes are large and fragile 50200 m using

9、 flow cytometry with larger internal diameter of the fluidics (150 to 250m). Easy to be brokencontrol Flow pressures to 510 psi, slow but not broken. Good for isolation.18精选课件Contaminants Stromal/vascular cells Aggregates of adipocytes Free lipid droplets Other debris Exclusion of Contaminants (4 st

10、eps)19精选课件 Collagenase Digestion Filtration Undigested tissue fragments are excluded. Centrifugation Separated from stromal/vascular cells Fragile Use rocking rather than vortexingPreparation of single cell suspension of free-floating adipocytes20精选课件Step 1. Initial separationby size Adipocytes (502

11、00 m) bigger than stromal/vascular cells(20 m) higher in Forward Scatter (FSC) The presence of highly refractile lipid droplets in adipocyte higher in SSC Overlap need further separation Small adipocytes ignored (entire analyze if necessary)21精选课件Contaminants Stromal/vascular cells Aggregates of adi

12、pocytes Free lipid droplets Other debris Exclusion of Contaminants (4 steps)22精选课件Step 2. 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, sp

13、end more time, W increased 23精选课件Step 2. Exclusion of aggregates and selection of singlets Stain nuclei by DyeCycle Violet (DCV) fluorescence. Single cells can be easily gated from aggregates & other debris This step couldnt exclude remaining stromal/vascular cells.24精选课件Step 3. Exclusion of single

14、stromal/vascular cells Stain the lipid droplets by LipidTOX Deep Red (LpdTX) Positive Selection of events containing lipid droplets25精选课件Step 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)Neg