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,BackgroundAim of the researchDifficulty S
2、olution,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,I
3、nvolved 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),Princ
4、iple 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 (CLSM),
5、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 adipocytes
6、 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 scannin
7、g 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 selectively
8、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 mEasy to be broken, when the flow rate (pressure) is large.,16,Difficulty,Adipocytes are large and fragile,502
9、00 mEasy to be broken, when the flow rate (pressure) is large.,Contaminants,Stromal/vascular cells Aggregates of adipocytes Free lipid dropletsOther debris,17,Solution,Adipocytes are large and fragile,50200 m using flow cytometry with larger internal diameter of the fluidics (150 to 250m).Easy to be
10、 brokencontrol Flow pressures to 510 psi, slow but not broken. Good for isolation.,18,Solution,Contaminants,Stromal/vascular cells Aggregates of adipocytes Free lipid dropletsOther debris Exclusion of Contaminants (4 steps),19,Solution,Collagenase DigestionFiltration Undigested tissue fragments are
11、excluded.Centrifugation Separated from stromal/vascular cellsFragile 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 in Forward Scatt
12、er (FSC)The presence of highly refractile lipid droplets 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 dropletsOther debris Exclusion of Contaminants (
13、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 WWhen aggregates(doublets) pass, spend more time, W increased,23,Solution,Step 2. Exclusion of ag
14、gregates and selection of singlets,Stain nuclei by DyeCycle Violet (DCV) fluorescence. Single cells can be easily gated from aggregates & other debrisThis step couldnt exclude remaining stromal/vascular cells.,24,Solution,Step 3. Exclusion of single stromal/vascular cells,Stain the lipid droplets by
15、 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
16、(phycoerythrin(PE)-conjugated) to stromal lineage markers are stained to exclude.(Negative selection),Negative selection,26,Solution,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 LI,
