1. detect and identify Bioanalytic Mithras LB 940 plate reader for research and screening HTRF & BRET applications Protein Interaction Networks, Nov. 2007, Paris

2. Bioanalytic Mithras LB 940

3. Bioanalytic Mithras LB 940 Berthold history & products TRF/HTRF basic settings BRET settings Mithras unique features TRF/HTRF further settings Other applications Good to know Questions

4. Bioanalytic 1949founded by Prof. Dr. Rudolf Berthold 1960 taken forward by Dr. Fritz Berthold 1989 purchased from EG&G Inc. USA (today: PerkinElmer Inc.) 2000re-purchased Dr. Fritz Berthold (& family) and Hans J. Oberhofer History

5. Bioanalytic BERTHOLD TECHNOLOGIES GmbH & Co. KG Calmbacher Straße 22 75323 Bad Wildbad, Germany www.Berthold.com/bio For more than 50 years in Bad Wildbad

6. Bioanalytic Instruments for Life Sciences Microplate Readers Imaging Systems Tube Luminometers Radio HPLC Monitors Gamma Counters

7. Bioanalytic Tube Luminometers

8. Bioanalytic NightOWL II LB 983 imaging systems

9. Bioanalytic NightHAWK LB 984 Gel doc HAWKview transilluminators

10. Bioanalytic HPLC Radio Monitor LB 513 Gamma Counter LB 2111

11. Bioanalytic Microplate Readers

12. Bioanalytic Microplate reader portfolio dedicatedmulti Absorbance☑☑ Luminescence☑☑ Fluorescence Intensity☑☑ Fluorescence Polarisation☑ Time-Resolved Fluorescence☑ AlphaScreen☑ BRET☑

13. Bioanalytic Mithras LB 940

14. Bioanalytic Mithras LB 940 Halogen excitation lamp Plate carrier excitation filters PMT & emission filters Flash excitation lamp

15. Bioanalytic Mithras LB 940 Waste outlet mains Reagent injectors Reagent tubings

16. Bioanalytic Applications & Settings

17. Bioanalytic Time-Resolved Fluorescence

18. Bioanalytic Time-Resolved Fluorescence Lanthanide Chelates Europium Samarium Terbium Dysprosium Excitation @ 320-340 nm

19. Bioanalytic Time-Resolved Fluorescence Long-lived fluor.: up to 1000 µs

20. Bioanalytic Time-Resolved Fluorescence Different emission wavelengths

21. Bioanalytic Time-Resolved Fluorescence Different emission decay times

22. Bioanalytic Time-Resolved Fluorescence Large Stoke´s shift

23. Bioanalytic Time-Resolved Fluorescence DELFIA® heterogeneous method: unbound labelled molecules must be washed away HTRF® (CisBio) homogeneous method: energy transfer between cryptate and acceptor

24. Bioanalytic TRF - DELFIA as immunoassay Signal after re-chelating w/ Enhancement solution Emission @ 615 nm

25. Bioanalytic LANCE: TR-FRET the homogeneous method (PKI) Eu-Chelate as donor (615 nm) APC, Cy5 or ULight as acceptor (665 nm)

26. Bioanalytic HTRF ® : TR-FRET the homogeneous method (CisBio) Eu-Cryptate as donor (620 nm) XL665 as acceptor (665 nm)

27. Bioanalytic Time-Resolved Fluorescence Xenon flash lamp broad-band excitation filter: cwl 320 nm confocal emission optics emission filter 615 nm (DELFIA Eu) emission filters 620 nm (Cryptate Eu) and 665 (acceptor e.g. XL665) gated PMT (red enhanced response for TR-FRET)

28. Bioanalytic TR-FRET performance Mithras typicaltarget S/N 620 nm (control/bkg)7040 cv of ratio 0 Std5 %10 % Delta F of Low FRET Std28 %15 % Delta F of High FRET Std900 %600 % CisBio reader evaluation kit (white 96 well half area)

29. Bioanalytic Label dialog TRF: DELFIA Counting time per well Always full lamp energy Emission filter Cycle time: 2000 Delay Time: 400 Reading Time: 400

30. Bioanalytic Label dialog TRF: HTRF Open default HTRF.par file or start from scratch Click Options | Read

31. Bioanalytic Label dialog TRF: HTRF Samples tab: Plate selection Measured wells Injected wells Reading order

32. Bioanalytic Label dialog TRF: HTRF Measurement tab: Operation settings Operation sequence Temperature settings

33. Bioanalytic Label dialog TRF: HTRF Counting time per well Always full lamp energy Emission filter Cycle time: 2000 Delay Time: 50 Reading Time: 300

34. Bioanalytic Label dialog TRF: HTRF

35. Bioanalytic Label dialog TRF: HTRF Export with “RawData Export Driver”

36. Bioanalytic Label dialog TRF: HTRF

37. Bioanalytic HTRF – further settings Ratio calculation & HTRF ratio calculation

38. Bioanalytic HTRF – further settings Ratio calculation & HTRF ratio calculation

39. Bioanalytic HTRF – further settings Calculated data are visible in Result pages

40. Bioanalytic HTRF – export of calculations Export with “Matrix Export Driver”

41. Bioanalytic HTRF – export of calculations

42. Bioanalytic Luminescence & BRET

43. Bioanalytic Luminescence & BRET Highly efficient fiber optics unsurpassed sensitivity in luminescence Possibility to insert filters enables sensitive BRET reading enables sensitive Chroma-Glo reading Unique aperture to reduce crosstalk 96, 384 and 1536 well plates

44. Bioanalytic Luminescence & BRET Performance < 6 amol ATP per well better than most dedicated luminometers Reading time minimum 0.05 sec per well Selected filter sets for BRET and BRET² as well as for Chroma-Glo

45. Bioanalytic Luminescence & BRET

46. Bioanalytic What is BRET ? Energy (light) of Renilla reaction is transferred to fluorescent protein Read-out at emission wavelength of fluorescent protein Occurs upon close proximity Occurs upon interaction of attached biomolecules

47. Bioanalytic 7 transmembrane receptor = G protein coupled receptor (GPCR) Renilla luciferase GPCR screening with BRET

48. Bioanalytic 7 transmembrane receptor = G protein coupled receptor (GPCR) R. luciferase Upon addition of the Renilla luciferase substrate coelenterazine blue light with a peak at 480 nm is emitted Coelenterazine 1 to 5 µM GPCR screening with BRET

49. Bioanalytic ligand / agonist Attachment of ligand causes activation of the receptor incl. dissociation of the G-protein subunits and phosphorylation of the receptor R. luciferase Coelenterazine 1 to 5 µM GPCR screening with BRET

50. Bioanalytic R. luciferase Coelenterazine 1 to 5 µM ß-arrestin/eYFP ß-arrestin/eYFP fusion protein attaches to activated GPCR; this results in proximity of the Renilla luciferase and the eYFP moiety, close enough for the emitted light of the luciferase reaction to cause an energy transfer upon which eYFP emits light at ist specific emission wavelength GPCR screening with BRET

51. Bioanalytic 10 5 COS cells per well transfected with receptor- Rluc (NK1 and ß 2 A resp.) and ß-arrestin-GFP vectors Agonist screening with Isopretenerol as positive control (5 wells) and ß 2 A receptor expressing cells GPCR screening with BRET

52. Bioanalytic Universal & functional for Gs, Gi and Gq Direct and signalling pathway independent Non-radioactive Ratio-metric Luminescence-based (no auto- fluorescence) Applicable for orphan receptors Short assay development (~ 3 weeks) More sensitive than IP 3 and Ca ++ GPCR screening with BRET

53. Bioanalytic BRET Leptin receptor assay End point assay Dose-response evaluation Agonist dispensed manually

54. Bioanalytic BRET Leptin receptor assay Selection of plate format Definition of reading order Selection of measured wells Selection of injectors #2 to be used for all wells for addition of substrate

55. Bioanalytic BRET Leptin receptor assay 1 dispense step 1 delay step 1 reading operation

56. Bioanalytic BRET Leptin receptor assay dispense step for adding Coelenterazine Inj. #2 30 µL Medium speed By well

57. Bioanalytic BRET Leptin receptor assay delay step allowing the Coelenterazine to enter the cells 5 s By well

58. Bioanalytic BRET Leptin receptor assay measurement 1 s 1 st emission filter 480 nm (Rluc signal) 2 nd emission filter 530 nm (YFP signal) By well

59. Bioanalytic BRET Leptin receptor assay

60. Bioanalytic BRET Leptin receptor assay

61. Bioanalytic BRET Leptin receptor assay

62. Bioanalytic BRET Leptin receptor assay

63. Bioanalytic BRET Leptin receptor assay Options | CurveFit dialog mBRET as source matrix Four Parameter as algorithm Logarithmic x and y scales Select or define units

64. Bioanalytic BRET Leptin receptor assay Options | CurveFit dialog Click Add Intercept FMI+(FMA-FMI)*0.5 is automatically inserted = 50 % line = IC50 Click Apply to all groups

65. Bioanalytic BRET Leptin receptor assay

66. Bioanalytic BRET Leptin receptor assay

67. Bioanalytic BRET Leptin receptor assay

68. Bioanalytic altern. Leptin rec. assay Alternative operation setup: End point assay Dose-response evaluation Agonist pre-dispensed in microplate Cells added with injector

69. Bioanalytic altern. Leptin rec. assay Selection of plate format Definition of reading order Selection of measured wells Selection of injector #2 to be used for all wells for addition of substrate Selection of injector #3 to be used for all wells for addition of cells

70. Bioanalytic altern. Leptin rec. assay 2 dispense steps 1 delay step 1 reading operation

71. Bioanalytic altern. Leptin rec. assay dispense step for adding cells Inj. #3 50 µL Low speed By plate

72. Bioanalytic altern. Leptin rec. assay dispense step for adding Coelenterazine Inj. #2 30 µL Medium speed By well

73. Bioanalytic altern. Leptin rec. assay delay step allowing the Coelenterazine to enter the cells 5 s By well

74. Bioanalytic altern. Leptin rec. assay measurement 1 s 1 st emission filter 480 nm (Rluc signal) 2 nd emission filter 530 nm (YFP signal) By well

75. Bioanalytic Kinetic BRET assay Repeated operation mode for kinetic reactions over several minutes 9 wells manual substrate and agonist addition

76. Bioanalytic Kinetic BRET assay Plate selection 9 wells to be measured

77. Bioanalytic Kinetic BRET assay 2 Luminescence operations Define number of plate repeats Ratio YFP/Rluc will automatically be calculated as LB4

78. Bioanalytic Kinetic BRET assay

79. Bioanalytic Kinetic BRET assay

80. Bioanalytic Kinetic BRET assay

81. Bioanalytic end of part 1

82. Bioanalytic Mithras – unique features DOPS – optical system Photomultipliers Crosstalk reduction Plate height adjustment JET Reagent injectors

83. Bioanalytic DOPS Unique optical design: Dedicated Optical Path System (DOPS)

84. Bioanalytic DOPS

85. Bioanalytic DOPS

86. Bioanalytic DOPS

87. Bioanalytic Photomultipliers Highest quality: Stringent selection Single Photon Counting

88. Bioanalytic PMTs - Selection Stringent selection procedure Low & stable background Blue/green/red efficiencies Long term stability At various temperatures

89. Bioanalytic PMTs - Selection

90. Bioanalytic PMTs – Photon Counting

91. Bioanalytic PMTs – Photon Counting Photon Counting Current Measurement 10 100 1000 10000 100000 1000000 10000000 1E-181E-171E-161E-151E-141E-131E-121E-111E-101E-091E-08 ATP [moles] Relative Light Units/s [RLU/s]

92. Bioanalytic PMTs – Current mode Light Level Output Result Low gain Mid gain High gain Switching points

93. Bioanalytic PMTs – Photon Counting Advantages of Photon Counting No need to calibrate No need to set HV range Single Dynamic range (no gain settings) More stable backgrounds Improved sensitivity Instrument is stable over years

94. Bioanalytic PMTs – Stability Mithras

95. Bioanalytic Cross-Talk Reduction Patented design to eliminate signals from not-measured wells Aperture wheel with 3 positions for 96, 384 and 1536 well sizes respectively Positioned automatically according to the plate format selected

96. Bioanalytic Cross-Talk Reduction

97. Bioanalytic Crosstalk (X-talk) spillover of light between wells constant signal S m signals in neighbour wells S d diagonal position S a adjacent position CT a = S a / S m CT d = S d / S m all signals background corrected

98. Bioanalytic Crosstalk Reduction

99. Bioanalytic Plate Height Adjustment Detects plate height automatically while plate is moved in Adjusts and minimizes „gap“ between plate surface and detection optics automatically No need to measure or inquire for plate height dimensions

100. Bioanalytic Reagent Injectors JET injection with bellow- type reagent injectors out in

101. Bioanalytic Reagent Injectors JET injection technology Located in reading positions 10 to 100 µl Injection into 384 well plates possible High precision & accuracy better 98%

102. Bioanalytic Reagent Injectors 300 msec

103. Bioanalytic Reagent Injectors

104. Bioanalytic Reagent Injectors

105. Bioanalytic Reagent Injectors Average precision: 99.67 % Average accuracy: 99.41 %

106. Bioanalytic Reagent Injectors Specific procedure for priming: „EXTENDED ECONOMY PRIME“ Efficient homogeneous filling Saving of reagents Incremental filling of the bellow combined with vibrations

107. Bioanalytic Reagent Injectors JET injector „dead volumes“ are actual working volumes, e.g. 1 mL in Centro syringe type dispensers need to flush at least 5 times their „dead volume“ to get homogeneous filling

108. Bioanalytic Reagent Injectors CV: 3.38 %

109. Bioanalytic Time-Resolved Fluorescence

110. Bioanalytic HTRF – calc. reader evaluat.

111. Bioanalytic HTRF – calc. reader evaluat.

112. Bioanalytic HTRF – calc. reader evaluat.

113. Bioanalytic HTRF – calc. reader evaluat.

114. Bioanalytic HTRF – calc. reader evaluat.

115. Bioanalytic HTRF – calc. reader evaluat.

116. Bioanalytic HTRF – calc. reader evaluat.

117. Bioanalytic HTRF – calc. reader evaluat.

118. Bioanalytic HTRF – calc. reader evaluat.

119. Bioanalytic HTRF – calc. reader evaluat.

120. Bioanalytic HTRF – calc. reader evaluat.

121. Bioanalytic HTRF – calc. reader evaluat.

122. Bioanalytic HTRF – calc. reader evaluat.

123. Bioanalytic Summary of Applications ApplicationRead-out Calcium MonitoringAequorin Luminescence Fura 2, Indo 1 Fluorescence Cell ViabilityATP/Luciferase Luminescence ToxicityResorufin Fluorescence ProteasesFluorescence TR-FRET Protein-protein interactionBRET AlphaScreen™ FRET / TR-FRET Protein quantificationNanoOrange Fluorescence Bradford Absorbance DNA quantificationPicoGreen Fluorescence

124. Bioanalytic Summary of Applications ApplicationRead-out ProliferationFormazan Absorbance ATP/Luciferase Luminescence Reporter GeneLuciferase Luminescence GFP Fluorescence KinasesFluorescence Polarisation Luminescence TRF / TR-FRET ImmunoassaysHRP Luminescence HRP Absorbance TRF / TR-FRET PhagocytosisLuminol Luminescence AmplexRed Fluorescence

125. Bioanalytic Summary of Applications ApplicationRead-out Functional GPCRBRET IP 3 TR-FRET cAMP AlphaScreen Ca ++ Luminescence Ca ++ Fluorescence CaspasesLuminescence Fluorescence TRF /TR-FRET Receptor-Ligand BindingFluorescence Polarisation TRF / TR-FRET NAD(P)HAbsorbance

126. Bioanalytic Increasing Throughput

127. Bioanalytic Increasing Throughput LB 930 BUTLER plate handling robot can be used with Centro and Twinkle, too two models: BUTLER 20 for 20 microplates BUTLER, universal base plate, 2 stackers, HTTL BUTLER 100 for 100 microplates BUTLER, universal base plate, 6 stackers, HTTL LB 931 Stacker unit 25 plate capacity 50 plate capacity

128. Bioanalytic Good to Know Selecting Filters Selecting Microplates Cleaning Injectors Priming Injectors 21 CFR part 11

129. Bioanalytic Selecting filters Does the filters’ transmission spectra fit with the label/fluorophore in question? Should the filter be broad or narrow? as a general rule, narrow filters should be used in photometric applications (absorbance measurements) otherwise, broad filters can/should be used with broad filters the background level can increase and destroy the results ! Is the filters’ blocking level good enough? the blocking level should be at least 10 -4

130. Bioanalytic FITC excitation spectra…

131. Bioanalytic FITC emission spectra…

132. Bioanalytic FITC excitation & emission

133. Bioanalytic Selecting filters Excitation and emission filter pairs are not to overlap with their transmission spectra!

134. Bioanalytic Selecting Filters Rule of thumb:  CWL >= 0.85 * (FWHM ex + FWHM em )

135. Bioanalytic Selecting filters Twinkle eliminates scattered light by second lens and aperture Angular light can pass interference filters -> cut-off shifted to lower wavelengths ! Select emission filter in Mithras with a CWL at even higher wavelengths missing in Mithras

136. Bioanalytic Selecting filters Fluorophores list

137. Bioanalytic Selecting microplates Absorbanceblack clear-bottom Fluorescenceblack Luminescencewhite BRETwhite Fluo. Polarisationblack Time-Res. Fluo.Black/white AlphaScreenwhite

138. Bioanalytic Selecting microplates Clear bottom microplates OK for absorbance Only when cell growth needs to be monitored before or after measurement Increase crosstalk (luminescence) Increase background due to light scatter (fluorescence)

139. Bioanalytic Selecting microplates Berthold plate descriptions

140. Bioanalytic Cleaning Injectors When? When changing reagents At the end of a measuring session After longer periods of non-activity Before starting a measuring session

141. Bioanalytic Cleaning Injectors How? Access the INSTRUMENT menu Select WASH Proceed as prompted

142. Bioanalytic Cleaning Injectors What to use? Solutions recommended by reagent manufacturer De-ionized water 40-70 % EtOH Mild acids or bases Berthold CLEANIT 45218

143. Bioanalytic Priming Injectors When? Before starting a measuring session When changing reagents

144. Bioanalytic Cleaning Injectors How? Access the INSTRUMENT menu Select PRIME Proceed as prompted

145. Bioanalytic Cleaning Injectors What to use? Prime lines with de-ionized water first Next, prime with reagents

146. Bioanalytic Kinetics mode “Kinetics” for short kinetics

147. Bioanalytic Kinetics mode “Repeated” for long kinetics

148. Bioanalytic Kinetics mode

149. Bioanalytic Kinetics mode Multiple calculation posibilities

150. Bioanalytic Mikrowin 21 CFR part 11 21 CFR part 11 compliancy - Advanced I or II ! controlled access system: user name & password audit trail: log file data files can be printed in human readable form data and parameter files can be protected user name and date/time stamps in data files documentation according to ISO 9001

151. Bioanalytic 21 CFR Users with password access

152. Bioanalytic 21 CFR Log file

153. Bioanalytic 21 CFR Printability of data files

154. Bioanalytic 21 CFR Protection of data and parameter files

155. Bioanalytic 21 CFR user name & date/time stamps in data files

156. Bioanalytic Questions?