BD Accuri C6 Flow Cytometer Overview. Continuous Measurement of Intracellular Calcium and Other Cellular Responses

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1 BD Accuri C6 Flow Cytometer Overview Maria Dinkelmann, PhD Senior Marketing Applications Specialist BD Biosciences Continuous Measurement of Intracellular Calcium and Other Cellular Responses Alfonso Blanco-Fernandez, PhD Director of Flow Cytometry University College Dublin

2 The BD Accuri C6 Flow Cytometer An affordable, full-featured, easy-to-use flow cytometer Two lasers and six detectors

3 The BD Accuri C6 flow cytometer Innovations in all the major components of a flow cytometer Fluidics: Peristaltic pumps and pulse dampeners allow an open fluidics system, miniaturization, and direct-volume measurement Optics: locked-down alignment Signal detection: broad dynamic range eliminates voltage adjustments Software: developed by high-tech anthropologists trained to facilitate humancomputer interactions

4 Sample Detector Fluidics Waste Laser Purge Flow Cell Laminar flow fluidics Patented pulse dampeners Non-pressurized, peristaltic pumpdriven system Volume measurement for absolute counts Minimum sample volume 50 µl if using a 1.5-mL conical tube Up to 10,000 events/second Sheath

5 Alignment and signal detection are optimized and locked down at manufacture 488-nm solid state laser SSC 640-nm diode laser PMTs for fluorescence detection FSC Diodes for scatter detection

6 Instrument-to-instrument variation is minimal Eight-peak data, multiple C6 instruments manufactured over a six-month period.

7 A robust and portable tool for challenging environments The Ecosystem Centre: Palmer Peninsula, Antarctica National Oceanic and Atmospheric Administration: Great Lakes Microcystis research project Lake Erie

8 Voltage adjustment 200 Slide courtesy of Dr. Alfonso Blanco-Fernandez, University College Dublin

9 Pre-optimized detectors: fixed voltage system Slide courtesy of Dr. Alfonso Blanco-Fernandez, University College Dublin

10 Intuitive software Sample Grid Cytometer Status Fluidics Controls Run Criteria Histograms Dot Plots Density Plots Analysis and Gating Tools Regions Quadrants Markers Real Time Updates Plot Statistics

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12 Continuous measurement of intracellular calcium and other cellular responses

13 Fluor. / a.u. Microscopy live cell imaging cells! Time (sec)

14 BD FACS440

15 Stop-flow method

16 Stop-flow method?

17 Stop-flow method BD FACStar Plus

18 Time Zero System (Cytek) Eric Chase & Harvey Schulte Keith Kelly BD FACStar BD FACStar Plus BD FACSVantage Coulter EPICs Coulter Elite

19 BD Accuri C6

20 Number Intensity

21 BD Accuri C6 Number Intensity

22 BD Accuri C

23 BD Accuri C

24 BD Accuri C6 Cliffs of Moher (Ireland)

25 BD Accuri C

26 BD Accuri C6

27 BD Accuri C6 Extremely important!! Check the State of the instrument (QC) Laser properly align and stable Instrument clean,...

28 BD Accuri C6 Peristaltic Pumps

29 BD Accuri C6 Gel loading tips

30 BD Accuri C6 How? Pay attention to the movie

31 488: 530/30: Fluo-4 488: 530/30: Fluo-4 488: 530/30: Fluo-4 488: 530/40: Fluo-4 488: 530/40: Fluo-4 488: 530/40: Fluo-4 Beckman Coulter Cyan ADP: A23187 EtOH A23187 Thapsigargin A23187 Accuri C6:

32 BD Accuri C6. Time limitations? Fluo-3 : 530/30 Fluo-3: 530/30

33 Air bubbles, any effect on the speed? Fluo-4 : 530/30 Cells Air Ionophore

34 Control on the addition time for all my samples?

35 Time between the addition and the analysis? 638 : 675/ : 675/25 Addition Start Point Analysis Start point

36 Calcium flux Fluo-4: 530/30 Ionophore EtOH EtOH EtOH

37 Calcium flux Fluo4 Adapted from RS. Lewis, The molecular choreography of a store-operated calcium channel, Nature, 2007.

38 Calcium flux Fluo4 Thapsigargin Adapted from RS. Lewis, The molecular choreography of a store-operated calcium channel, Nature, 2007.

39 Calcium flux 2-APB Fluo4 Thapsigargin Adapted from RS. Lewis, The molecular choreography of a store-operated calcium channel, Nature, 2007.

40 488: 585/40: Fluo-4 488: 585/40: Fluo-4 488: 585/40: Fluo-4 488: 585/40: Fluo-4 488: 585/40: Fluo-4 488: 585/40: Fluo-4 Calcium flux Ionophore Thapsigargin Ionophore 2-APB Ionophore Thapsigargin

41 488 : 530/30: Fluo-4 Fragmented cells

42 488: 530/30: Fluo-4

43 488: 585/40: Fluo-4 488: 585/40: Fluo-4 488: 585/40: Fluo-4 488: 585/40: Fluo-4 488: 585/40: Fluo-4 488: 585/40: Fluo-4 Calcium flux Ionophore Thapsigargin Ionophore 2-APB Ionophore Thapsigargin

44 Calcium flux 488: 585/40: Fluo-4 Osmotic pressure?

45 How do FSC vs SSC change because of osmotic pressure? Erytrocytes lyse with dh 2 O

46 How do FSC vs SSC change because of osmotic pressure? Erytrocytes lyse with dh 2 O Beads

47 488: 585/40: Fluo-4 488: 585/40: Fluo-4 488: 585/40: Fluo-4 488: 585/40: Fluo-4 488: 585/40: Fluo-4 488: 585/40: Fluo-4 Calcium flux Osmotic pressure??? Ionophore Thapsigargin Ionophore 2-APB Thapsigargin Ionophore

48 Some calcium profiles

49 WT mcherry Some calcium profiles in mcherry transfected cell lines 488: 585/40: Fluo-4 488: 585/40: Fluo-4 488: 675LP mcherry 488: 675LP mcherry 488: 585/40: Fluo-4 488: 585/40: Fluo-4 488: 585/40: Fluo-4 488: 585/40: Fluo-4

50 Some calcium profiles in mcherry transfected cell lines

51 Some calcium profiles in mcherry transfected cell lines Drug Drug Drug

52 Some calcium profiles in mcherry transfected cell lines

53 Nanoparticles uptake :530/30: Yellow-Green npart Time (min)

54 Nanoparticles uptake 488: 530/30: Yellow - Green NPs NPs

55 Nanoparticles uptake

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61 What if my sample has to be at 4ºC?

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63 Nanoparticles uptake

64 Nanoparticles uptake

65 Nanoparticles uptake

66 Nanoparticles uptake

67 Nanoparticles uptake Fixed cell in suspension covered by nanoparticles, after incubation at 4 C

68 Nanoparticles uptake

69 Algae physiological analysis Kinetics of 4 different algae (FCS Express v.4)

70 Algae physiological analysis Kinetics of 4 different algae (FCS Express v.4) Addition time point (2:00 min)

71 Algae physiological analysis

72 Algae physiological analysis Illumination?

73 Algae physiological analysis

74 Conclusions: We have been able to establish a good method for a continuous measurement of morphological and intracellular calcium changes and other cellular responses

75 Acknowledge: Gethin McBean Alice Vine Monica Gorin Kristina Gegenbauer Nadezhda Glezeva Anna Lesniak Liam Brennan

76 Acknowledge: Bob Penney Clare Rogers Jack Ball Kate Easten Grant Howes Leo J. Ostruszka Stuart Bradley

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