用流式細(xì)胞儀分析脂肪細(xì)胞

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:10.10

2、16/B978-0-12-411619-1.00015-X.,2,,CONTENTS,,BackgroundAim of the researchDifficulty Solution,Background,What are adipocytes (fat cells)?Major cells in the adipose tissue.,3,Background,Lipid droplet,Two kinds of adip

3、ocytes in human,4,Prospects of adipocyte biology,Background,Lipid store,Inhibit eating Increase metabolism,,Leptin,5,Prospects of adipocyte biology,Background,Adipokines,Involved in energy metabolism,Lipid store,6,Prosp

4、ects 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,

5、,Background,Flow Cytometry (FCM),Principle diagram,9,,Background,Volume,Morphological complexity of cells, Total DNA/RNA content, Apoptosis, Cell viability, ... ... ... ...,10,Aim of the research,A me

6、thod for analysis and isolation free-floating adipocytes by Flow Cytometry.,,Analysis,Flow cytometry (FCM)Confocal laser scanning microscope (CLSM),,Isolation,Fluorescence-activated cell sorting (FACS),11,Aim of the res

7、earch,,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

8、free-floating adipocytes 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-fl

9、oating adipocytes by Flow Cytometry.,Aim of the research,,Analysis,Flow cytometry (FCM)Confocal laser scanning microscope (CLSM),Imaging Flow Cytometer(Amnis),12 Images for every cell,Quantitive,High resolution imaging,

10、,,,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 selectively add different charge, fall

11、ing into different containers.,15,A method for analysis and isolation free-floating adipocytes by Flow Cytometry.,Difficulty,Adipocytes are large and fragile,50–200 μmEasy to be broken, when the flow rate (pressure) is

12、large.,,16,Difficulty,Adipocytes are large and fragile,50–200 μmEasy to be broken, when the flow rate (pressure) is large.,,Contaminants,Stromal/vascular cells Aggregates of adipocytes Free lipid dropletsOther debris

13、,,17,Solution,Adipocytes are large and fragile,50–200 μm ——using flow cytometry with larger internal diameter of the fluidics (150 to 250μm).Easy to be broken——control Flow pressures to 5~10 psi, slow but not broken. Go

14、od for isolation.,,18,Solution,Contaminants,Stromal/vascular cells Aggregates of adipocytes Free lipid dropletsOther debris ——Exclusion of Contaminants (4 steps),,19,Solution,Coll

15、agenase —— DigestionFiltration —— Undigested tissue fragments are excluded.Centrifugation ——Separated from stromal/vascular cellsFragile —— Use rocking rather than vortexing,Preparation of single cell suspension of fr

16、ee-floating adipocytes,,20,Solution,Step 1. Initial separation——by size,Adipocytes (50–200 μm) bigger than stromal/vascular cells(<20 μm) — higher in Forward Scatter (FSC)The presence of highly refractile lipid dropl

17、ets in adipocyte— higher in SSCOverlap —— need further separationSmall adipocytes—— ignored (entire analyze if necessary),,21,Solution,Contaminants,Stromal/vascular cells Aggregates of adipocytes Free lipid drop

18、letsOther debris ——Exclusion 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,

19、 Width — passing time(size), Area= H x WWhen aggregates(doublets) pass, spend more time, W increased,,23,Solution,Step 2. Exclusion of aggregates and selection of singlets,Stain nuclei by DyeCycle Violet (DCV) fluoresc

20、ence. Single cells can be easily gated from aggregates & other debrisThis step couldn’t exclude remaining stromal/vascular cells.,,24,Solution,Step 3. Exclusion of single stromal/vascular cells,Stain the lipid dro

21、plets 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 th

22、e free lipid droplets.The remaining stromal contaminants bearing the fluorescent antibodies (phycoerythrin(PE)-conjugated) to stromal lineage markers are stained to exclude.(Negative selection),,Negative selection,26,So

23、lution,Validation,qRT-PCRStromal cells marker (such as CD34)Black--before flow sorting (High expressed)White--after flow sorting (Almost undetectable),,27,,Analysis and Isolation of Adipocytes by Flow Cytometry,Yang