1536-well FLIPR:A Possibility or Probability? Or, How I spent last week! Gary Allenby AstraZeneca Lead Generation, Charnwood, U.K.

Transcription

1 1536-well FLIPR:A Possibility or Probability? Or, How I spent last week! Gary Allenby AstraZeneca Lead Generation, Charnwood, U.K.

2 Background

3 Introduction Why 1536? Pro s and Con s Pro s Cost of agonist is decreased: Five fold saving in agonist cost (384 well plate = 10ul into 40ul) (1536 well plate = 2ul into 8ul) Agonist plate volume can be a limitation in automation (384 well): In 384 well, 100ul agonist plate 10ul) = 8 or 9 test plates (3456 wells) In 1536 well, 100ul agonist plate 2ul) = 11 test plates (16896 wells):4.8 times more wells Logistics of plates and volumes of liquid are significantly less savings in storage space, plate handling and loading, and waste stream generated:e.g. 200,000 data points = Con s Plate costs increase: Approx two to three fold more expensive Perceived difficulty is increased

4 Comparison of Methodogies Methodology Cell # per well grown o C/5%CO 2 Aspirate media 384 well 5,000 (50ul) EMBLA well 1,500 (5ul) EMBLA 1536 (leave 2ul) Dispense loading buffer * Incubate Multidrop (35ul) o C Flexispense or Aquamax 1536 (5ul) o C Dispense compound (pre-diluted DMSO) Incubate Platemateplus (5ul 0.5% [final]) o C Platemateplus or Cybiwell (1ul 0.5% [final]) o C FLIPR dispense and read 10 ul of agonist (EC 80 = 5nM); 4 x 2 then, 60 x 1 second read 2 ul of agonist (EC 80 = 5nM); 4 x 2 then, 60 x 1 second read * Loading buffer = HBSS containing; Hepes (20mM), Probenecid, brilliant black (0.5mM) and Fluo-4 (2uM)

5 Plate types that work (and don t work)! 384 well normal Greiner plates 384 well low volume 1536 well hibase 1536 well lo-base

6 Which plate works? Incident angle of light hitting the bottom of the hi-base plate casts a shadow on the first row of the plate 384 well bottom 1536 well hi-base bottom 1536 well lo-base bottom Recommendation:1536 lo-base plate from Greiner

7 Do cells grow in 1536 well plates? 384 well (5,000 cells, 50ul/well) 1536 well (1,500 cells, 5ul/well) Cells grown for 24 hours in Greiner black, clear bottom 384 or lo-base 1536 well plates with lids in a humidified Heraeus BB6220 incubator at 37oC

8 Media removal MDC (Skatron 1536 washer) Uses 2 x 384 well pins to aspirate the plate in 2 passes Only used to aspirate (media) Pro s Reasonably Fast complete 1536 in ~15 secs Con s Depth of Greiner lo-base plate is greater than the length of the needles, therefore leave media behind (2ul) Requires tweeking to get the plate carrier level with the plate

9 Cells (5ul) and Fluo-4+BB (5ul) dispensing 1536 well Aquamax Pro s Fast complete 1536 in ~5 secs Accurate Dispense 1ul (8%CV) Can also Dispense to 384 square well plates Con s Large dead volume Small ish pressurised reagent vessel (250 ml) Reasonably good instrument for small volume (1 to 10ul)

10 Cells (5ul) and Fluo-4+BB (5ul) dispensing Flexispense Pro s Reasonably Fast complete 1536 in ~15 secs Accurate Dispense 0.5ul (6%CV) Can also Dispense to 384 square well plates Con s Requires tweeking Dead volume Reasonably good instrument for small volume (1 to 10ul) 5ul dispense of Fluo-4 + BB to all wells (serpentine) Manifold designed in-house

11 Compound addition (DMSO) Add 1ul/well of pre-diluted DMSO (0.5% final) Platemateplus or Cybio Cybiwell 384 disposable tips One wash with 3 cycles

12 Assay performed in 384 well plates Example of positive control, whole plate data and Z-factor Z factors generated from 100 plates positive controls negative controls

13 FLIPR 1536 Hardware issue - tips 10ul Packard tips on the CCS head 1536 well lo-base plate with every other well removed

14 1536 well FLIPR Setup Tuned the laser at 0.6 W Imaged the 1536 yellow.fcf plate (calibration plate) plate to overlay the 1536 mask Calibrated the instrument using the calibration plate Imaged the calibration plate to determine variability of illumination Ran experiments from 384 well Matrix polypropylene source plate to Greiner lo-base 1536 well plate to quadrant 1 0.6W, 0.4 sec exposure, 4 x 2sec reads (baseline) plus 40 x 1 sec read after addition 2ul addition of agonist to 8ul in well (2ul media + 5ul Fluo-4 containing brilliant black [0.5mM], 1ul pre-diluted DMSO [0.5%])

15 1536 well FLIPR Software - calibration

16 1536 well FLIPR Software alignment of mask and plate map These values work for Greiner lo-base plate Best to change this values for dispensing heights

17 1536 Well Data on FLIPR

18 1536 well FLIPR Data 1 st quadrant Some negative controls respond needs further work Average loading fluorescence was 8237 (SD was 2060)

19 1536 well FLIPR Data 1 st quadrant Less negative controls respond probably due to pipettor height (4mm) Average loading fluorescence was (SD was 2340)

20 1536 well FLIPR Data - all quadrants Some negative controls respond needs further work Average loading fluorescence was 9922 (SD was 2115) Do not ask for this option yet; it is troublesome at best and requires additional work on software and hardware to make it work properly

21 1536 well FLIPR Data all quadrants Reasonable consistency Some negative controls respond needs further work Average loading fluorescence was (SD was 2652)

22 1536 well FLIPR Data suggests that 1536 well FLIPR assays are possible on a FLIPR 384

23 WHY 1536 well FLIPR? I have just about solved my 384 well problems, why do 1536? Test more compounds? Reduce screening costs 1 million 384 = $50,000 for ligand 1 million 1536 = $10,000 for ligand Logistics are decreased (number of plates) Cell number is decreased therefore less cell culture Decreased compound utilisation? Provide higher quality data (replicates) as; Quadruplicate (data mining, pharmacophore searching, virtual screening etc processes all require high quality data) 2 concentrations (10 and 1uM) Single 4 concentrations (10, 3, 1, 0.3uM)

24 1536 well FLIPR The Future Since FLIPR 384 works, FLIPR 3 should work better (camera sensitivity) Software needs work Hardware tips!! The most robust approach is to replace with needles with adequate washing Matrix tips a possibly if these can be registered with the head and clamped on? Molecular Devices what s in the pipeline? A 384 with needles? A 1536 (needles)?

25 Thanks go to: James Longden Kathy Dodgson