Pharmacology Facilities

In Vitro Pharmacology Laboratories

Several laboratories at ANSTO are designed to carry out in vitro pharmacological investigations. These labs are equipped with instruments to enable the extractions of receptors of interest from live cells or fresh/frozen animal tissues (-80oC freezers, tissue homogenisers, centrifuges); instruments to investigate the mass action and functional roles of drug-receptor complexes (cell harvesters, Beta Liquid Scintillation counters) both at cellular and subcellular levels. The labs are capable of handling radioactive work such as autoradiography, radioimmunoassays and cytotoxicity, from a wide range of radioisotopes depending on their half lives such as ¹²³I, ¹²⁵I, ³H, ³⁵S and ³²P.

The labs are also equipped with instruments for other cell-related work such as UV spectrophotometers and multi plate readers.

In Vivo Laboratories

Assessment of the in vivo pharmacokinetics of our novel radiotracers is performed in a dedicated laboratory, equipped with the latest SPECT/CT imaging equipment and biodistribution facilities as well as a team of biologists who perform the in vivo evaluations.

Gamma Counter

Evaluating the biodistribution of our radiotracers in vivo is a critical element in our drug development. We use gamma counting as a means to obtain specific organ uptake and excretion profiles of our radiotracers at specific time points. Gamma counting is also used in vitro binding assays.

Cell culture

We culture a number of tumour cell lines (e.g. prostate, melanoma, breast and glioma) to support both in vitro and in vivo studies. We can learn a great deal about our new pharmaceuticals by studying their effects in cells. For example, we can determine whether the radiopharmaceutical will be taken up by tumour cells or if the pharmaceutical can induce cell death. In order to study novel radiopharmaceuticals in an animal model of cancer, we culture cells for tumour inoculations into rats and mice.

Flow cytometry

The Beckman Coulter Cell Lab Quanta SC is a 5 parameter (volume, side scatter and 3 colour fluorescence) flow cytometer. The instrument has two light sources; a mercury arc lamp and a 488nm solid state laser. The instrument is capable of accurately measuring the size and the granularity of a cell while measuring up to three fluorescent dyes simultaneously.

Real time PCR

PCR is the amplification of a DNA sequence using enzymatic replication. The process, developed by Kary Mullis in 1983, has become an important tool in both medicine and biology.

DNA replication has three distinct steps dissociation, annealing and extension. A thermal cycler is used to perform and repeat these three steps for up to 40 cycles. One cycle consists of first dissociating the two strands of DNA by heating, the temperature is then cooled in the annealing step to allow primers to bind and finally the temperature is adjusted to the running temp of a thermal stable DNA polymerase. This binds to the double stranded primer bound sites and replicates the missing strand 5’ to 3’. Every cycle the amount of target sequence is doubled, resulting in the exponential amplification of the target DNA.

ANSTO uses an iQ5 Real Time PCR thermocylcer from Bio-Rad. Real time PCR involves the addition of a data acquisition step whereby the amount of DNA or a target sequence is quantified by measuring the relative fluorescent units resulting from DNA binding dyes or sequence specific fluorescence quenching probes, both of which are in use at RRI.

ANSTO uses this PCR facility for looking at the expression of target proteins by measuring the amount of transcript present in a cell or tissue. This involves the addition an extra reverse transcription step in which complimentary DNA copies are made of the total RNA in a tissue and the target cDNA of the transcript of interest is measured by amplification. Using this technique we can characterise the expression of a target receptor between tissues as well as observe the up or down regulation of transcription due to drug treatments.

Histology

Our histology laboratory is equipped with a cryostat, microtome, paraffin embedding station as well as lght and fluorescent microscopes. We can perform various types of histological and immunocytochemical staining.

Autoradiography

We are equipped with facilities to perform autoradiographic experiments (in vitro and in vivo) using various radioligands. Our facilities include, small and large cryostats, radioligand binding laboratory, dark room facilities for film development, phosphorimager and image analysis systems (MCID, Image-Pro) for quantification of autoradiographs.

Cyromicrotome

Used for sectioning large specimens for whole body autoradiogrphy.

Phosphorimager

Uses special resuable imaging plates to create an image of the radioactivity present in a sample. These plates are more sensitive than X-ray film and therefore allow shorter exposure time.