Microplate readers

A very versatile piece of equipment that is affordable for individual laboratories is the microplate reader. This allows multiple samples to be analyzed at once, commonly in a 96-well format, although 384- and 1536-well formats are available. Typical measurements that can be performed include UV-Vis absorbance, fluorescence, or luminescence, allowing a range of assays to be performed, such as cell growth, enzyme kinetics, enzyme stability, or enzyme-linked immunosorbent assay [60-62]. Functionality can be increased by the use of liquid dispensing systems or automatic plate handling. 

Cells are lysed for Firefly and Renilla luciferase assays using the Dual-Luciferase Reporter Assay system (Promega), following the manufacturer s instructions. We use a multimode microplate reader with automatic injectors (FLUOROstar Optima from BMG Labtech, OfFenburg, Germany) for luminescence measurements. 

Budantsev, A. Yu. and Budantseva, T.A. (2005). Photometric analysis of paper tests using microplate readers. Journal of Analytical Chemistry (Russsian) 60 794-797. 

Measure the absorbance of all wells using a microplate reader with a filter set at 410nm. 

The foregoing method has been adapted by Davalos and others (2004) using a conventional fluorescence microplate reader and applied to pure compounds (benzoic and cinnamic acids and aldehydes, flavonoids, and butylated hydroxyanisole) and to wines, as well as to commercial dietary antioxidant supplements. Eberhardt and others (2005) have also proposed a similar method for the determination of the antioxidant activity in broccoli. 

Spectrophotometric plate readers Perkin-Elmer s lambda reader, an automated microprocessor-controlled, microplate reader, offers the flexibility of configuring a reliable, user-friendly, versatile system, capable of accommodating a wide variety of assays requiring calorimetric measurement on microscale (<300pl) samples. These assays include ELISA, protein determination, cytotoxicity, cytoproliferation and antibody sensitivity testing. 

Lambda reader automated microplate reader Perkin-Elmer Corporation Analytical Instruments Division 761 Main Avenue, Norwalk Connecticut 06859-0012 USA... 

Cell inhibition rate analysis Cells were seeded in 96-well plates at a density of 1.5/ 104 cells/well in quadruplicate and incubated at 37°C in a 5% C02 atmosphere. After 24 hr, cells were treated with 0.1% DMSO (control) or, compound at various concentrations (0.01 - 0.33 mM) for 48 hr. Cell inhibition rate was analyzed after 48 hr using the Cell Counting Kit CCK-8 (Dojindo Laboratories, Japan), according to the manufacturer s instructions. OD values were measured at a wavelength of 450 nm using a Multi-Detection Microplate-Reader (Bio-TEK, Winooski, VT). 

Lorenzen, A. and Kermedy, S.W. 1993, A fluorescence-based protein assay for use with a microplate reader. Anal. Biochem. 214 346-348. 

Leave the fu-st column of wells blank to zero the microplate reader. Fill the wells with PBS only. 

Free radical scavenging activity was assessed using the DPPH (1, 1-di-phenyl-2-picrylhydrazyl) assay as described by Harbilas et al. [22] with incubation time increased to 65 min. Briefly, 250 tiL of 100 timol/L DPPH dissolved in methanol was added to 40 p.L of extract (tested at 5 concentrations) in a microplate well. A standard curve of ascorbic acid (positive control) was included as a reference and all data were blanked against a treatment with only methanol. Absorbance was read with a microplate reader at 517 nm. The inhibitory concentration for 50% scavenging (IC50) of each extract was calculated and compared to the IC50 of the ascorbic acid standard curve. 

IL-4 2 0.14 HRP colorimetric Microplate reader 96-well plastic Pierce Endogen... 

Heltweg, B. and Jung, M. (2002) A microplate reader-based nonisotopic histone deacetylase activity assay. Analytical Biochemistry, 302, 175-183. 

The absorbance was measured on a microplate reader (Tecan, Switzerland). 

Add 25 pL of Cell Titer Glo to all white solid-bottom plates. Incubate the plate at room temperature for at least 1 h on a rotating platform. Read luminescence using a luminescence microplate reader (see Note 12). 

Fig. 12. Effect of spontaneous apoptosis on the activation of caspase-3 in human eosinophils. Eosinophils (2 x 10 /ml) were cultured for 3 h. Cells were lysed and caspase-3 activity was measured by caspase-3 colorimetric assay kit (R D Systems). Enzymatic products were measured at 405 nm with BIOTEK EL340 ELISA microplate reader (BIO-TEK Instrument Inc., Vermont). Human recombinant caspase-3 (5 U) (Calbiochem, California) was used as a positive control. The stimulation index was determined by direct comparison to the level of the normal control. Background readings from cell lysates and buffers were substracted from the readings of both induced and uninduced samples samples before calculating the stimulation index in caspase-3 activity. The differences between control and treated groups were assessed by Student s i-test. P < 0.05 P < 0.001 (Z2).

Spectrofluorometers and microplate readers measure the average properties of bulk (pL to mL) samples... 

Spectra MAX microplate reader (Molecular Devices, Sunnyvale, CA, USA). 

Measure absorbance at 405 nm using a microplate reader (rrr Note 4). 

Measure the time-course of luminescence for several minutes using microplate reader. 

For the spectrophotometric method, there is no sample workup, allowing one to run -4 assays/hr. This can be increased to -16 to 100 or more samples/hr depending on equipment features and automation, such as multiple cuvette holders/changers and 96-well microplate readers. For the colorimetric procedure, sample workup requires -10 min/subsample, but several samples can be batch processed simulta-... 

For this purpose, Bruiser has already coupled the microplate stacking device Twister 1 to its microplate reader [22]. In this combination, which is controlled by OPUS software, 40 IR microplates can be measured automatically. To load samples with high throughput, the Microlab 4000 SP autosampler can be used. Both formats (96 and 384) of the Bruker silicon microplates are suitable for automatic loading of various types of samples (proteins, cells, culture media). 

Spectrophotometer any suitable microplate reader able to measure absorbance at the appropriate wavelength. For TMB, this is 450 nm, having stopped the reaction with 0.5 M H2S04. 

Syngas, including high level C02 under pressurized conditions. The out gas was bubbled into water to absorb formed MeOH. The MeOH concentration in each solution was determined by the addition of inorganic dye to the solution and the absorbance was determined with a microplate reader (reproduced by permission of The Japan Petroleum Institute from 34]). 

The dynamic range of the TRF-ELISA covers IgG concentrations from 5 to lOOng/mL [108]. Detection limits of more than one magnitude lower (0.1 ng/mL) can be obtained with commercially available time-resolving microplate readers. 

The method may also be used the other way round with GOx as the enzyme label, glucose as substrate, and [Eu(Tc)] as the fluorescent indicator. In that type of assay, the presence of enzyme-labeled antibodies is indicated by an increase in the observed fluorescence intensity. It should be noted that commercially available fluorescence microplate readers, preferable equipped with time resolution, are also suited for the screening of all the microwell plate-based assays presented in this chapter. Nevertheless, the imaging process is much faster, accomplished in the order of one second, and enables ratiometric measurements. 

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