Extracellular vesicle cytometry: Technical challenges and solutions. May 11, 2017 Chris Spring, Research Core Facilities

Transcription

1 Extracellular vesicle cytometry: Technical challenges and solutions May 11, 2017 Chris Spring, Research Core Facilities

2 A little nomenclature Oncosomes Ectosomes Microvesicle Microparticle Argosome Cardiosome Archeosome Dexosome Texosome Tolerosome Prostasome Prominosome Calcifying matrix vesicle Cellular and Molecular Life Sciences, August 2011, Volume 68, Issue 16, pp

3 Learning Objectives: Use technical knowledge to define challenges in EV cytometry Pre-analytical (sample prep) Analytical issues and controls Partial solutions to challenges in Sensitivity Gating Analysis

4 Let s get some things straight Flow Cytometry Fluid flow Cell Measurement Flow Evometry?

5 Preanalytical preparation Anticoagulant type (spontaneous EV generation; citrate) Samples stay vertical Fasting where possible (chylomicrons) Discard first collection tube, remove tourniquet in case of plasma analysis JEV :29260 Yuana Yuana et al.

6 Preanalytical preparation Collection and spin processing- under 1 hour 300xg spins (2) + 800xg spin (render acellular FCM to confirm) Some spin faster (1550xg) JEV :29260 Yuana Yuana et al.

7 Preanalytical preparation If you must freeze, flash freeze in LN 2 Single freeze thaw and storage up to 1 year OK Does increase PS - profiles on EVs JEV :29260 Yuana Yuana et al.

8 Component analysis: Challenges RNA contents- eliminate RNA from serum protein complexes, lipoprotein complexes UC then density gradient to remove higher density protein aggregates Washing and free antibody/dye Immunoaffinity UC may be able to remove lipoproteins but also poor EV recovery, EV aggregation Sepharose columns (SEC- lipoprotein removal)

9 Sounds EASY! Let s do it!

10 Pulse Signals: Area Height and Width Cell Laser beam Pulse Signal = Area Height Width

11 Fluorescence quantitation: Use of area 10um bead 1000 U FITC 100um bead 1000 U FITC

12 Height versus Area Pulse Signal Laser beam (exaggerated) Area Height Width

13 Competitive Dodgeball Easy! Hmmmm Throw more balls (get a really big laser) Aw, FACS

14 Effects of Laser power

15 Controls Filtration (removal) Detergent sensitive Detergent (SDS/Triton/NP-40) lysis to eliminate false positives Filtration of antibodies sometimes necessary Swarm detection- dilutions and/or fluorescence labeling Low flow rates/pressure Annexin V is NOT a pan-specific EV marker!

16 Swarm and ICs Leukocyte 700nm EV 200nm EV

17 EVs: Mie think this is getting tricky Cell (microns) 500nm X = nm X = 1.28 EV (100s of nanometers)

18 Fluorescence and scatter: Smaller particles diffract more evenly more around the particle ( FSC and SSC) Smaller particles have much smaller antigen density

19 Refractive Index Higher RI = more scattered light Some typical RIs: Polystyrene Silica nm PS Urinary EV (fresh) Urinary EV (lyophilized) blood EV platelet EV J Extracell Vesicles :25361

20 Problem: Gating and Standards Nano erythrosomes Exometry.com Rosetta calibration AJP Lung, August 2016, Volume 310, Issue 9, pp L Eur J Pharm Sci. September 2016 (16)

21 The problems (during flow) 1) swarm 2) Light must be scattered (issue of both size and RI) 3) Scatter or fluorescence must be detected by PMT (QE) 2 ) fluorescence emissions required (how much fluorophore is required to be seen? Sample FP/FN) These things are affected by the wavelength of the light we are using

22 Let s use a different LASER!

23 Wavelength and Light Scatter: A way forward? FSC FSC FSC SSC and fluorescence SSC and fluorescence

24 R9220 PMT sensitivity Mie Scatter 500nm X = nm X = 1.28 Rayleigh Scatter

25 405nm SSC increases signal ~2 fold

26 405nm SSC increases signal ~2 fold

27 Biological EVs resolved at 405nm SSC

28 Take Home Messages Method of isolating EV is crucial Depends on EV type, biological sample type, etc Challenges of detection regardless of approach Key controls (swarm, ICs, etc) Violet rules, blue drools

29 OMG he s finally going to stop talking!!!