用流式细胞仪分析脂肪细胞.ppt

1、Analysis and Isolation of Adipocytes by Flow Cytometry,Yang LI 2016-04-05,Majka 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.,2,CONTENTS,Background Aim of the research Difficulty

2、 Solution,Background,What are adipocytes (fat cells)?Major cells in the adipose tissue.,3,Background,Lipid droplet,Two kinds of adipocytes in human,4,Prospects of adipocyte biology,Background,Lipid store,Inhibit eating Increase metabolism,Leptin,5,Prospects of adipocyte biology,Background,Adipokines

3、,Involved in energy metabolism,Lipid store,6,Prospects of adipocyte biologySpecifically from adipose tissue,Background,Lipid store,Adipokines,Energy metabolism,7,Prospects of adipocyte biologyAdipokines play an significant role in biological function.,Background,8,Background,Flow Cytometry (FCM),Pri

4、nciple diagram,9,Background,Volume, Morphological complexity of cells, Total DNA/RNA content, Apoptosis, Cell viability, . . . .,10,Aim of the research,A method for analysis and isolation free-floating adipocytes by Flow Cytometry.,Analysis,Flow cytometry (FCM) Confocal laser scanning microscope (CL

5、SM),Isolation,Fluorescence-activated cell sorting (FACS),11,Aim of the research,Analysis,Flow cytometry (FCM) Confocal laser scanning microscope (CLSM),Beckman Coulter-MoFlo XDP,Analysis rate:100,000 events/sec. Sort rate: 70,000 events/sec.,12,A method for analysis and isolation free-floating adipo

6、cytes by Flow Cytometry.,Aim of the research,Analysis,Flow cytometry (FCM) Confocal laser scanning microscope (CLSM),Quantitive,High resolution imaging,13,A method for analysis and isolation free-floating adipocytes by Flow Cytometry.,Aim of the research,Analysis,Flow cytometry (FCM) Confocal laser

7、scanning microscope (CLSM),Imaging Flow Cytometer(Amnis),12 Images for every cell,Quantitive,High resolution imaging,14,A method for analysis and isolation free-floating adipocytes by Flow Cytometry.,Aim of the research,Isolation,Fluorescence-activated cell sorting (FACS),A passing cell can be selec

8、tively add different charge, falling into different containers.,15,A method for analysis and isolation free-floating adipocytes by Flow Cytometry.,Difficulty,Adipocytes are large and fragile,50200 m Easy to be broken, when the flow rate (pressure) is large.,16,Difficulty,Adipocytes are large and fra

9、gile,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,Solution,Adipocytes are large and fragile,50200 m using flow cytometry with larger internal diameter of the fluidics (150 to 250m).

10、 Easy to be brokencontrol Flow pressures to 510 psi, slow but not broken. Good for isolation.,18,Solution,Contaminants,Stromal/vascular cells Aggregates of adipocytes Free lipid droplets Other debris Exclusion of Contaminants (4 steps),19,Solution,Collagenase Digestion Filtration Undigested tissue f

11、ragments are excluded. Centrifugation Separated from stromal/vascular cells Fragile Use rocking rather than vortexing,Preparation of single cell suspension of free-floating adipocytes,20,Solution,Step 1. Initial separationby size,Adipocytes (50200 m) bigger than stromal/vascular cells(20 m) higher i

12、n 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,Solution,Contaminants,Stromal/vascular cells Aggregates of adipocytes Free lipid droplets Other debris Exclusio

13、n of Contaminants (4 steps),22,Solution,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, spend more time, W increased,23,Solution,Ste

14、p 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,Solution,Step 3. Exclusion of single stromal/vascular cells,Stain

15、the lipid droplets by LipidTOX Deep Red (LpdTX) Positive Selection of events containing lipid droplets,25,Solution,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)