BD BioCoat and BD Falcon FluoroBlok Tips and Techniques. Jeff Partridge, M.S. April 27, 2010

Transcription

1 BD BioCoat and BD Falcon FluoroBlok Tips and Techniques Jeff Partridge, M.S. April 27, 2010

2 Overview Part I Part II Part III BD FluoroBlok System Description Tips and Tricks Questions and Answers 2

3 What is the BD Falcon FluoroBlok Insert System? Insert (individual or multiwell) Apical chamber Pore BD FluoroBlok membrane Basal chamber Base plate Cross section of an insert system not to scale 3

4 What is the BD Falcon FluoroBlok Insert System? Light-tight PET membrane Dyed membrane blocks light from nm while allowing for multiplex detection assays Choice of membrane pore sizes 24 individual cell culture inserts or 24- and 96-multiwell insert plates available in 3 or 8 µm pore sizes Run homogeneous assays in real time with non-destructive detection Rapid data collection without the need for plate washing or manual cell scraping and counting Determine migration or invasion of cells in real time Quantitative detection Use of fluorescence detection 8 μm pores visible light 4

5 What Can You do with the BD FluoroBlok Insert System? Cell Migration Haptotaxis movement in response to a gradient of adhesion sites or ECMs Chemotaxis movement toward a chemical gradient Cell motility chemokinesis, cell movement Cell Invasion Degradation of a physical barrier Pore-occluding BD Matrigel Matrix-coated BD FluoroBlok Insert Systems Co-culture Multiple cell types in close proximity A variety of cell types on either BD Falcon or BD BioCoat FluoroBlok Insert Systems can be used. 5 *

6 Insert System Handling pipettes and plates A repeating pipettor is recommended for the 24-multiwell or individual insert system. A multi-channel pipettor is required for the 96-multiwell insert system. The 96 square-well plate must be kept level to minimize siphoning. When adding cells to inserts, pipette gently so as not to disrupt the matrix. Add somewhat slowly, instead of quickly adding directly onto the matrix: this is somewhat easier when using 96-well insert systems. 6

7 Insert System Handling insert volumes Volumes added to apical and basal chambers must match. Typical chamber volumes apical basal nominal range nominal range 24-well 250 μl μl 750 μl μl 96-well 50 μl μl 200 μl μl 7

8 Insert System Handling thawing and rehydration of matrix For BD Matrigel matrix-coated inserts, remove the foil bag from -20 C and allow it to reach RT. Once thawed, you must use all of that insert system. For five packs, you may reseal the package and use the unthawed systems. Buffering system is important. Rehydration medium must match conditions: medium use CO 2 incubator PBS best to use non-co 2 incubator *carbonate/bicarbonate-buffered medium would require CO 2. No need to rehydrate uncoated membranes. 8

9 Insert System Handling cells Use good aseptic technique. Use low passage # cells. Do not use cells that are over-confluent to start. Use cells that are in log-growth phase for best results. 9

10 Cell Culture Insert Handling individual inserts + individual inserts (w/flanges) companion plate (w/notches) = insert flanges resting in plate notches *When using individual cell culture inserts, both the inserts and companion plates must be used. 10

11 Insert System Handling individual inserts and multiwells Be careful when feeding or aspirating individual inserts. Gently move aside insert; try to keep or replace flanges in notches. Be extra careful when removing medium from 96-well insert system fill ports. 11

12 Insert System Handling the bubble effect Bubbles should be eliminated at all steps. Chemoattractant should be added to the bottom chamber via the access port. To minimize bubbles, add to the apical chamber then to the basal chamber. bubble under insert will influence reading cells may not migrate or stain = bad 12 *

13 What Format Should I Use? platform choice and scalability Wide choice of formats: Cell Culture Inserts (clear) Multiwell Insert Systems (clear) BD FluoroBlok cell culture inserts BD FluoroBlok multiwell inserts 24-Multiwell Insert System 96-Multiwell Insert System Tumor Invasion System (BD Matrigel matrix-coated) 24 Individual Cell Culture Inserts (clear) BD Matrigel matrix invasion chamber 24 Multiwell Tumor Invasion System 96-Multiwell Tumor Invasion System Angiogenesis System: Endothelial Cell Migration (human fibronectin coated) 24 Individual Cell Culture Inserts (clear) 24 BD FluoroBlok cell culture inserts 24 Multiwell Insert System 96-Multiwell Insert System 13

14 What Pore Size Should I Use? platform choice and scalability Application Pore size Transport and Permeability 0.4 µm, 1 µm Drug transport across epithelial cell monolayer (i.e. Caco-2) Co-culture and cell monolayer 0.4 µm, 1 µm, 3 µm, 8 µm Cell-cell paracrine interactions Human endothelial monolayer permeability Marine phytoplankters In vitro model systems 0.4 µm, 1 µm, 3 µm Human epidermal model Human urothelium model Blood brain barrier Cell Migration 3 µm, 8 µm Leukocytes Endothelial cells Fibroblasts Neutrophils Tumor Cells Osteoblasts Smooth muscle cells PBMCs Cell Invasion 3 µm, 8 µm Tumor Cells Fibroblasts Epithelial cells Note: This is not an exhaustive list of all cell types and applications. The appropriate pore size is cell type and application specific. 14 *

15 Insert System Use assay setup Ensure that you know where your controls are and what they should be: chemoattractant {serum, VEGF, etc.} no chemoattractant no cells drug vehicle stain only No one setup is better than another as long as you know where your samples and controls are. test 1 control test test test 2 control test 3 control control control control control control control test 4 control 15

16 Insert System Use calculation of invasion % Calculation examples migration represents 100% of cells coming thru the membrane can compare invasion as a fraction of that 100% Why? not all cells will necessarily pass through the insert. Other examples can also use increase over a control. Image Reference Partridge, J., Flaherty, P. (2009). An In vitro FluoroBlok Tumor Invasion Assay. JoVE doi: /

17 Insert System Use screening example How many replicates do you need? We can use Z to assist. Two replicates should be suitable for screening in this system. Tumor Invasion System - Z' values 96 multiwell Tumor Invasion System - Z' values 24 multiwell 1.00 average Z' = average Z' = Z' 0.50 Z' replicate number replicate number 17 *

18 BD FluoroBlok Inserts Do You Need an ECM Coating? Extracellular matrix (ECM) coating may be needed by your cells. Some cells require ECMs for attachment or signaling. Choice of ECM and coating conditions needs to be optimized: time, temperature? for immediate use or to be stored and used later? 18

19 BD FluoroBlok Inserts self-coating tips Consider choice of buffers. Pore occluding BD Matrigel Matrix layer Consider coating volume and thickness. Swirl coating solution in well. Be sure when coating you remove all bubbles. BD FluoroBlok membrane, 8 µm pore size Reconsider fresh coat then use vs. make and store for later use. Consider the use of BD BioCoat Inserts. The underside of membranes can be coated. 19

20 Osteoclast Migration Toward ECM Substrates self-coating example FLG 29.1 cell line, human preoc, induced to differentiate by TPA Underside of 8 µm inserts coated with ECM substrates at 20 µg/ml Spessotto P., et al., Journal of Cell Biology, 158(6):1133 (2002). 20 *

21 BD FluoroBlok Insert System cell labeling dyes Any fluorescent dye derived from the fluorescein, rhodamine and cyanine families can be used with this system. The emission wavelength must be between nm. your dye here Ultraviolet-inducible dyes tend to be incompatible with the BD Falcon FluoroBlok insert since they tend to emit light in the blue range. Can I multiplex? Yes! with proper separation between the dyes. Spectrum image from under GNU free documentation license. 21

22 BD FluoroBlok Insert System dyes 22

23 BD FluoroBlok Insert System dyes 23

24 BD FluoroBlok Insert System multiplexing interneurons granule cells interneurons granule cells 1 μm pores visible with some DAPI stained nuclei (blue) that have migrated thru pores Cells plated on top of insert (A,E) Migrating neurons (B,C,F,G) MAP2 stained somata and dendrites (red) DAPI stained nuclei (blue) Hassoun, A., et al., J Neurosci. Methods. 166(2): (2007). 24

25 BD FluoroBlok Inserts detection instruments You will need: A fluorescent plate reader with bottom-reading capability -AND- An inverted fluorescent microscope for confirmation and troubleshooting -OR- A fluorescent imager, i.e., BD Pathway Bioimager or Cellomics HCS ArrayScan BD Pathway 855 High-Content Bioimager 25

26 BD FluoroBlok Inserts detection instruments Plate readers compatible with BD FluoroBlok insert systems include: BioTek Synergy (4, 2, HT), FLx800 BMG LABTECH PHERAStar (Plus), OPTIMA, POLARStar/FLUOStar (Omega, Galaxy) MDS Analytical Technologies SpectraMax readers (Multimode M5/M5 e, M2 e ; Gemini EM) PerkinElmer EnVision, Victor series, HTS Tecan Safire 2, GENios, SpectraFluor Plus Thermo Scientific Cellomics HCS ArrayScan Fluoroskan Ascent and many more! 26

27 BD FluoroBlok Inserts detection instruments Plate readers not 100% compatible with BD FluoroBlok insert systems include: MDS FlexStation II can only be used if the carrier device is removed 24-Multiwell format only MDS Analyst HT and Analyst GT are compatible with the 96-Multiwell format not the 24-Multiwell format, as the door is too short PerkinElmer Fusion beam size is too large for 24-well plates autofluorescence 27

28 BD FluoroBlok Inserts plate reader set-up Confirm the plate map supplied with the reader or input the correct coordinates and well diameter into the plate map. Make sure the correct diameter is used: 6.5 mm for 24-well and 3.2 mm for 96-well. Plate reader set-up guide 28

29 BD FluoroBlok Inserts plate reader set-up Single or multiple reads per well? for 24-multiwell, multiple reads are generally available single read focuses on center of the membrane multiple reads use reader-specific pattern (within-well average) overall CV improves with multiple reads, however it will take longer to read a plate Read areas from Tecan SAFIRE 2 (24-multiwell) Y-line X-line 6 6 square 7 7 ring 7 7 circle 29

30 BD FluoroBlok Inserts plate reader tip Ensure each plate is properly placed into the plate carriage at the A1 position. A1 plate carriage FBM insert 30 *

31 Cell Labeling Methods What should you use? Pre-Labeling labeling cells in vitro prior to assay Post-labeling labeling cells on the underside of membrane following migration or invasion Intrinsically-labeled cells expressing Green Fluorescent Protein or analogs (e.g., transfected) Underside of membrane showing SYTO24 (nuclear stain) labeled HT-1080 cells (green) which have migrated. Pores (yellow) are also visible. 31

32 BD FluoroBlok Inserts how to pre-label cells Generic pre-label protocol Stain cells when they are attached to flask Add dye at appropriate concentration for min Rinse monolayer with DPBS Remove cells as usual (trypsin) Wash 2X then perform assay DiIC 12 (3) labeled HT-1080 cells (red) which have invaded (left) or migrated (right) through Tumor Invasion System. 32 *

33 BD FluoroBlok Inserts kinetic assays Q: How do I run a kinetic assay? A: You need to pre-label and either take multiple manual readings or use a plate reader that has both temperature and atmospheric capabilities. Two approaches: HT-1080 and 3T3 pre-labeled with 5 µg/ml DiIC12(3) and plated in a BD BioCoat tumor invasion system Assay was run for 24 hours in a standard incubator with readings taken at various time points PC3-GFP cells were pre-labeled with SYTO 82, plated in BD FluoroBlok invasion chambers (selfcoated BD Matrigel matrix) Real-time analysis of tumor cell invasion with a FLUOstar OPTIMA plate reader enclosed in an AtmosBag (5% CO 2 ), reader set at 37 C * Partridge, J. et al., Society of Biomolecular Screening 2005 (conf.). Quantitative and High-throughput Screening of Tumor Cell Invasion Using a Cell-based model. *BMG Labtech Application Note 144 (12/2006) 33

34 BD FluoroBlok Inserts kinetic assays kinetic assay % invasion HT T time (h) Standard incubator, read manually Fig. 3. Using AtmosBag, automated read* Fig. 4. Standard incubator, read manually* Partridge, J. et al., Society of Biomolecular Screening 2005 (conf.). Quantitative and High-throughput Screening of Tumor Cell Invasion Using a Cell-based model. *BMG Labtech Application Note 144 (12/2006) 34

35 BD FluoroBlok Inserts kinetic assays and optimization Fold increase over control THP Time (mins) Fold increase over control Monocytes Time of incubation (mins) Time course of MCP-1 induced chemotaxis in THP-1 and monocytes Pre-labeled cells in the inserts were incubated with 25 nm MCP-1 in the bottom chamber Bottom fluorescence was measured at varying time points Data are means ±SD from a typical experiment (n=4 wells) BD Biosciences Technical Bulletin # *

36 BD FluoroBlok Inserts tips drug studies DMSO can be used without adverse effect at concentrations up to 2%. Some drug compounds adhere to plastics: you could use BD Gentest enhanced recovery plates for 24-multiwell and 96-multiwell inserts. Understand the compounds you are using: solubility will it remain in solution? at what ph? Do you need to add drug to both apical and basal chambers? RFU DMSO has no significant effect on Tumor Cell Invasion 2% 1% 0.50% 0.25% 0.13% 0% [DMSO] HT1080 3T3 doxycycline IC 50 (μm) invasion migration Post-label Pre-label RCFP labeling method did not effect IC *

37 BD FluoroBlok Inserts high-content screening assay HUVEC in 24-multiwell Angiogenesis System 24-multiwell insert system 96-multiwell insert system better signal-to-background ratio using nuclear stain / HCS than calcein AM labeling / plate reader EC well 96-well AAL nm 25 nm data in line with published results Mastyugin, V., et al., J Biomol Screen. 9(8): (2004). 37 *

38 BD FluoroBlok Inserts co-culture egfp-10t1/2 cells added to apical side; HUVEC seeded on base plate NOS inhibitor (L-NMMA) reduced migration of mural cell precursors toward HUVEC Satoshi Kashiwagi, et al., J. Clin. Invest. 115: (2005). 38 *

39 BD FluoroBlok Inserts use of non-adherent cells neutrophil chemotaxis 10,000 8,000 Control Chemoattractant RFU 6,000 4,000 2, hours time (h) 1.5 hours Pre-labeled for 15 minutes in flask Cells migrate through and then fall from insert and collect beneath insert 39 *

40 Some Selected Q&A why isn t my assay working? Q: Why isn t my assay working? A: Low invasive cells donor effect (see next slide) Q: What can I do? A: Try pre-label to get kinetic data increase chemoattractant concentration (5% 10%) increase invasion time (24h 72h or more) increase cell density (should try curve initially) typical seeding density 24-well: 25,000-50,000 cells/well 96-well: 10,000-20,000 cells/well you may need to optimize stain concentration you might need to use a different chemoattractant 40

41 BD FluoroBlok Inserts donor variability Human MSCs pre-labeled with CellTracker Green working [fmlp] established Human MSCs from different donors pre-labeled with CellTracker Green 100 nm fmlp as chemoattractant Anand Viswanathan, et al.,stem Cells 25: (2007). 41

42 BD FluoroBlok Inserts assay optimization Neuron migration thru BD Matrigel matrix in response to motogen (BDNF) Hassoun, A., et al., J Neurosci. Methods. 166(2): (2007). 42

43 BD FluoroBlok Inserts assay optimization tips Effect of MMP inhibitor 1'10' Phenanthroline on HMVEC Invasion 2000 HUVEC Migration HFN coated vs. uncoated Fluorescent Units Control VEGF(4ng/ml) 0.1ug/ml 1ug/ml 10ug/ml 20ug/ml Fluoresence Intensity HFN Coated Uncoated VEGF(4ng/ml)+ 1'10' Phenathroline [VEGF] (ng/ml) BD Matrigel matrix-coated BD FluoroBlok 3 µm pore size, 24 multiwell insert Optimized for endothelial cell invasion Human fibronectin-coated BD FluoroBlok 3 µm pore size, 24- and 96-multiwell inserts Optimized for endothelial cell migration 43 *

44 BD FluoroBlok Inserts how to test the system Acquire 3 or 8 µm BD FluoroBlok insert system in the format of your choice. Label HUVEC (3 µm) or HT-1080 (8 µm) in a TC flask with 5 µg/ml of calcein AM / HBSS [1 h, 37 C, 5% CO 2 ]. Wash the cells and seed in basal medium. Add 5% FBS in basal medium as the chemoattractant. Incubate for 4 hours at 37 C, 5% CO 2. Read the insert system on your plate reader. Confirm by observing the cells on the membrane using an inverted fluorescent microscope. 44 *

45 Selected Q&A removal of cells Q: Can cells be removed from the underside of membrane if attached? A: Yes, use trypsin or cell scraping. Try your standard protocol based on cell type and conditions. 45

46 Selected Q&A condensation Q: Will condensation on the lid affect my experiment or the system? A: No. This is a bottom-reading system. 46 *

47 Additional Thanks to... Bill Fiore Paula Flaherty Kevin Kelly Amyntrah Maxwell Shabana Islam Amy Laws 47 *

48 Upcoming Webinar May 12, 2010 In Vitro Models for Studying Angiogenesis 48 *

49 Questions? Technical Support: In the U.S. tel: Outside the U.S. Visit bdbiosciences.com/webinars For research use only. Not intended for use in diagnostic or therapeutic procedures. BD, BD Logo, and all other trademarks are the property of Becton, Dickinson and Company BD 49

50 Selected References general tumor invasion protocol Partridge, J., Flaherty, P. (2009). An In vitro FluoroBlok Tumor Invasion Assay. JoVE doi: /1475 kinetic invasion assay, multiplexing *BMG Labtech Application Note 144 (12/2006) invasion/migration drug screening (doxycycline) BD Biosciences Technical Bulletins #441 and 442. HCS screening of migration assay Mastyugin, V., et al., J Biomol Screen. 9(8): (2004). neuronal motogen screening Hassoun, A., et al., J Neurosci. Methods. 166(2): (2007). Time Course of MCP-1 Induced Chemotaxis in THP-1 and Primary Monocytes BD Biosciences Technical Bulletin #457 DiI Labeled Osteoclast Migration Toward ECM Substrates Spessotto P., et al., Journal of Cell Biology, 158(6):1133 (2002). NO-Mediated Migration Satoshi Kashiwagi, et al., J. Clin. Invest. 115: (2005). 50

51 Selected References Chemotaxis of Undifferentiated and Transfected HL-60 cells using 3 µm BD FluoroBlok Inserts Christophe, et al., J. Biol. Chem. 276:21585 (2001). Human MSC Chemotaxis Anand Viswanathan, et al.,stem Cells 25: (2007). Effects of Eph B4 Receptor Activation Primary Human Microvascular Endothelial Cells Jena J. Steinle, et al., J. Biol. Chem. 277(46):43830 (2002). A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays Zhang, et al., J Biomol. Screen 4:67 (1999). CD155 (Polio Virus Receptor) plays a key role in cell motility during tumor cell invasion and migration Sloan, et al., BMC Cancer, 4:73 (2004). Chemotaxis Study: To Determine Therapeutic Potential of SDF-1 in Hematopoietic Stem Cell Mobilization Pelus, et al., Letters in Drug Design & Discovery, 1:126 (2004). 51