Microsoft word - an_infinite_200_pro_gcm_v1 0.doc

Infinite® 200 PRO – Gas Control Module (GCM™) Enabling long-term cell-based assays with eukaryotic cells via controlled CO2 partial pressure The investigation of biological processes requires control that maintains a certain atmospheric CO2 level time-dependent analysis of cellular signals, from several hours (ranging from 3 to 10 %), to avoid pH shifts which would up to several days. However, performing long-term readouts prevent proliferation or cause cell death (1). with living eukaryotic cells inside common microplate readers has time limitations. This technical note describes the Most microplate readers currently available offer temperature capability of Tecan’s Infinite 200 PRO multimode reader to control but lack the ability to control the CO2 level inside the regulate the CO2 partial pressure inside its measurement measurement chamber. Therefore, long-term studies that chamber. The patent pending Gas Control Module (GCM) require cell incubation combined with in-between enables long-term cell-based kinetic measurements inside the measurements (real-time kinetic) cannot be performed inside reader, without negatively effecting cell proliferation and the reader. Periodic transfer of the microplate from the CO2 incubator to the microplate reader can present difficulties if performed manually, and may result in the data being Proliferation and survival of eukaryotic cells is strictly distorted by overnight gaps where measurements could not be dependent on the environmental temperature and on the taken. Automated robotic systems eliminate this problem, but culture medium conditions (1). The pH of most cell culture media is typically controlled by a bicarbonate buffer system and a precisely controlled atmospheric CO2 partial pressure The Infinite 200 PRO GCM (Figure 1) has been developed to that stabilizes the buffer system. Common cell culture control the CO2 partial pressure within the reader’s incubators are therefore equipped with a sensor-based CO2 measurement chamber, from 0 to 10 %, enabling long-term studies with eukaryotic cells using alternating incubation and measurement steps. Cellular survival and proliferation is therefore no longer limited to common CO2 incubators, and long-term cell-based analysis, such as cell proliferation Cell culture and test set-up
studies, can be performed inside the reader, offering Human squamous epidermoid carcinoma cells (A431), stably reproducible measurements without gaps in the data. transfected with EGFP, were grown to confluence in DMEM high glucose supplemented with L-glutamine, sodium pyruvate, penicillin / streptomycin, HEPES and 5 % heat-inactivated fetal calf serum (FCS) at 37 °C and 5 % CO2 in a humidified atmosphere (standard CO2 incubator with passive humidity control, Forma Steri-Cycle 371, Thermo). The cells were harvested using trypsin / EDTA, then resuspended in fresh growth medium containing 5 % FCS, seeded into black Lumox, 96-well tissue culture plates (5000 cells / well in 200 µl filling volume) and covered with a standard microplate lid (Figure 2). Figure 1 Tecan’s patent pending Gas Control Module (GCM), compatible with the Infinite 200 PRO series. Instrument
Figure 2 Plate layout; 5000 cells were seeded with and without FCS. ● Infinite M200 PRO Quad4 Monochromators™-based multimode reader, including Gas Control Module (GCM) After overnight incubation (~14 hrs) in a standard CO incubator, the culture medium was replaced (rows A-D were filled with fresh DMEM containing 5 % FCS, rows E-H were Microplates
filled with fresh DMEM without FCS) and all plates were ● 96-well, Lumox™, black with transparent bottom, tissue sealed by covering the interspace between plate and lid with Parafilm® (Brand GmbH, Germany) to avoid evaporation during the long-term growth study. Alternatively, plates can be Reagents
sealed using a real-time PCR film (e.g. ThermalSeal ● Human squamous epidermoid carcinoma cells (A431, possible, as Lumox plates enable gas transfer through their ● Dulbecco's Modified Eagle Medium high glucose (DMEM), heat-inactivated fetal calf serum (FCS, PAA ● Enhanced green fluorescent protein (EGFP) proliferation was determined using three different ● penicillin / streptomycin (PAA Laboratories) I. GCM plate
All plates were measured with 0 µs lag time and 20 µs Plate left in the Infinite M200 PRO reader integration time using the GRE96fb.pdfx plate definition file, with active CO2 control for incubation and and all recorded fluorescence signal intensities were calculated relative to the initial fluorescent signal (initial cell number at t = 0 hrs, representing 100 % GFP signal). Figure 3 shows growth curves of cells seeded at an initial concentration of 5000 cells / well in DMEM containing 5 % 535 (20) nm Pre-optimized for max. cell number / well FCS (A) and DMEM without FCS (B), respectively. Table 1 Handling and measurement parameters for microplate incubation Cells incubated with 5 % FCS
and measurement in the Infinite M200 PRO with GCM. Figure 3A shows growth curves of cells cultured in DMEM with 5 % FCS. Cells incubated and measured in an II. Positive control plate
Infinite M200 PRO with CO2 control (green circle) show Plate incubated in a standard CO2 incubator and manually transferred to significant proliferation up to 75 hrs (700 %, ~ 3.5 x 104 cells). This is comparable to the growth of the positive control (red triangle), with 900 % proliferation (~ 4.5 x 104). Growth curves from the positive control experiment lack data points due to overnight breaks, whereas growth curves from the long-term kinetic measurement performed in the Infinite M200 PRO with GCM do not have this critical limitation. Cells incubated and measured in a microplate reader without CO2 control (blue square) show limited proliferation up to 18 Pre-optimized for max. cell number / well hrs (140 %, ~7 x 103 cells). After this period, cells stop proliferating and the signal actually decreases down to 75 % Table 2 Handling and measurement parameters for microplate incubation in a standard CO2 incubator and measurement in the Infinite M200 PRO. III. Negative control plate
Cells incubated without FCS
Figure 3B shows growth curves of cells cultured in DMEM reader without CO2 control for incubation without FCS. This is relevant for many applications where FCS in the medium is disadvantageous. For example, FCS is regularly omitted when incubating cells with a compound that may induce cytotoxicity, as it non-specifically binds to the compound, partly inhibiting its uptake by cells. As expected, cells grown in medium without FCS show significantly lower overall proliferation rates compared to cells grown in medium containing 5 % FCS. Cell proliferation of the Pre-optimized for max. cell number / well positive control (330 %, 1.65 x 104 cells) was comparable to Table 3 Handling and measurement parameters for microplate incubation proliferation of cells incubated and measured in the and measurement in the Infinite M200 PRO without GCM. Infinite 200 PRO with GCM (260 %, 1.3 x 104). Again, cells incubated and measured in a reader without GCM show non- significant proliferation up to 18 hrs (135 %, ~ 6.75 x 103 cells) which then declines to 75 % (3.75 x 103 cells). Results presented in this technical note clearly demonstrate that the Infinite 200 PRO multimode reader, combined with Tecan’s new Gas Control Module, offers the capability to perform long-term cell-based studies lasting several days. Over the whole 75 hr period, cells left in a reader with GCM proliferate comparably to cells left in a common CO2 incubator, whereas cells left in a reader without CO2 control stop proliferation after several hours. Growth curves resulting from experiments performed in an Infinite M200 PRO with GCM do not lack any data points (no overnight gaps), which is a significant benefit for many long-term experiments. Tecan’s new patent pending Gas Control Module is an innovative solution for cell-based experiments, offering rigorous environmental control within the detection chamber. By offering precise regulation of carbon dioxide levels within the reader, the GCM provides a more stable culture environment over time, making it ideally suited for real-time analysis of biological processes. This integrated gas inlet with external control of CO2 stabilizes the pH value of the culture medium, helping to improve cell growth. Figure 3 Proliferation of cells cultured with (A) and without FCS (B) and To investigate possible evaporation during long-term incubation, the remaining filling volume was determined. For the plate incubated in the standard CO2 incubator, which was equipped with passive humidity control, an average volume of 190 µl (i.e. 95 %) remained per microplate well. For the plates incubated in Infinite M200 PRO readers (with and without GCM) an average volume of 180 µl (i.e. 90 %) remained after a period of 75 hrs. Similar growth is seen in the reader with GCM compared to the data of the standard cell incubator if sealed plates with gas permeable bottoms are used, demonstrating that humidity control is not mandatory inside (1) Martin Clynes, Animal Cell Culture Techniques, Springer Lab Manual, 618 pages, ISBN-10: 3540630082, 1998 We would like to express our thanks to Univ.-Doz. Dr. Kristjan Plaetzer and Verena Ziegler, Mag. Rer. Nat. (Division of Physics and Biophysics, Department of Materials Science and Physics, University of Salzburg) for providing the cell cultures and performing the experiments. Human squamous epithelial carcinoma cells 2-(4-(2-Hydroxyethyl)-1-piperazinyl)-ethanesulfonic acid Austria +43 62 46 89 33 Belgium +32 15 42 13 19 China +86 21 2898 6333 Denmark +45 70 23 44 50 France +33 4 72 76 04 80 Germany +49 79 51 94 170
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Tecan Group Ltd. makes every effort to include accurate and up-to-date information within this publication, however, it is possible that omissions or errors might have occurred. Tecan Group Ltd. cannot, therefore, make any representations or warranties, expressed or implied, as to the accuracy or completeness of the information provided in this publication. Changes in this publication can be made at any time without notice. All mentioned trademarks are protected by law. For technical details and detailed procedures of the specifications provided in this document please contact your Tecan representative. This brochure may contain reference to applications and products which are not available in all markets. Please check with your local sales representative. Tecan and Infinite are registered trademarks and Quad4 Monochromators and GCM are trademarks of Tecan Group Ltd., Männedorf, Switzerland. Lumox is a trademark of In Vitro Systems & Services, Germany. Parafilm is a registered trademark of American Can Company, USA. ThermalSeal RT2RR is a trademark of Excel Scientific, USA.
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