Analysis of prostate tumour cell invasion using BD FluoroBlok™ and a BMG LABTECH microplate reader

Introduction

The mechanism of prostate metastasis to bone marrow is a complex multistage process that is only beginning to be understood. Initial steps include down regulation of molecular binding complexes and production of enzymes such as matrix metallo-proteinases leading to the loss of cell to cell adhesion within the tumour and extravasation of tumour cells. Once in the peripheral blood the circulating prostate tumour cells bind specifically to and invade through the bone marrow endothelial barrier and basement membrane, leading to formation of bone metastases.

Traditional invasion systems are time consuming with each assay involving washing, scraping with a cotton bud to remove non-invading cells, staining, drying and manual counting. Most importantly the assay is inflexible and limited in that the inserts can only present the researcher with a fixed end point. The BD FluoroBlok^TM tumour cell invasion system is a high throughput alternative. The system is versatile and sensitive, involving no manipulation of inserts, thus reducing labour and time. It allows the researcher to do multiple real-time readings of the same inserts before and after introducing stimuli. The inserts are made of a dyed PET membrane that has a wide fluorescence blocking range from 490-700 nm allowing a range of potential fluorophores to be used. The system can be automated with the incorporation of the BMG LABTECH microplate reader, to increase efficiency and high throughput of assays for drug discovery, cell migration, motility and chemotaxis studies.

Principle

Materials & Methods

All components of the tumour cell invasion system were purchased from BD Biosciences.

• HTS FluoroBlok^TM Insert 24 well / 8 μM pores
• 24 well Companion Plate for use with 9mm pores
• BD Matrigel^TM Matrix 5ml
• BD BioCoat^TM Tumour Cell Invasion System 
• 24-Multiwell Insert Plate 8 μM pre coated Matrigel^TM
• BMG LABTECH microplate reader with fluorescence detection and either Gas Vent Connection or Atmospheric Control Unit (ACU) for gas control

All tissue culture reagents and SYTO 82 were purchased from Invitrogen.

The FluoroBlok^TM tumour cell invasion system was prepared as shown in Figure 2. All cultures were set up using phenol red free RPMI 1640 media. FluoroBlok^TM cell culture inserts were coated with 100 μl of Matrigel^TM (diluted at 1:25 with RPMI 1640) and allowed to set for 2hr at 37°C (Matrigel^TM pre-coated FluoroBlok^TM inserts can be used).

Coated inserts were placed in the 24 well companion plate containing 1ml of RPMI 1640 / 0.1% BSA with or without chemoattractant. In the top half of the insert 2x10⁵ GFP or SYTO 82 labelled PC-3 cells were plated in 0.25 ml of RPMI 1640 / 0.1 % BSA. Plates were then incubated at 37°C for 18 hr to allow invasion to occur after which a final end point reading was taken on the BMG LABTECH plate reader. For real time invasion, the plate was placed in the BMG LABTECH reader pre warmed to 37°C and 5% CO₂ was inflated either with the ACU or via Gas Vent. The BMG LABTECH reader was set to take readings every hour for 21 hr.

Results & Discussion

Initially, a comparison was made between the manual counting and the automated counting system of the FluoroBlok^TMinvasion chambers. Two identical assays were set up and counted manually or by the BMG LABTECH microplate reader. Both systems gave similar results (not shown). 

Initially a comparison was made between the manual counting and the automated counting system of the FluoroBlok^TM invasion chambers. Two identical assays were set up and counted manually or by the BMG LABTECH microplate reader. Both systems gave similar results (not shown).

Cells expressing a fluorescent protein are the ideal for use in these applications as no further manipulation is needed to be able to receive a signal when reading in the BMG LABTECH microplate reader. However, not all cells can be tranfected easily, especially primary cells. There are a number of alternatives to overcome this by either pre-labelling or post-labelling cells. Earlier investigations demonstrated that post-labelling was too unreliable hence pre-labelling was the method of choice. The problems inherent with prelabelling were the risk that the dyes could affect cell function and invasion. For this reason; the need for optimization of labelling time and side effects needed to be established beforehand. Pre-labelling is advantageous because it allows real-time invasion studies. The first step was to test whether the BMG LABTECH plate reader was able to support cells over 21 hours to gain real-time invasion data. PC3-GFP cells were pre-labelled with SYTO 82 and plated in FluoroBlok^TM invasion chambers. These were placed in the BMG LABTECH reader with either ACU or Gas Vent set at 37°C overnight infl ated with 5% CO₂ in air and set to take readings every hour of both GFP (488/520 nm) and SYTO 82 (530/570nm) for 21 hr. Figures 3 and 4 show that the BMG LABTECH reader supports the FluoroBlok^TM invasion chamber assays over a period of 21 hr; showing similar data to an experiment where invasion chambers were left in an incubator (37°C / 5% CO2 in air) overnight and read at specific times.

Conclusion

Using a BMG LABTECH microplate reader with integrated Gas Vent or Atmospheric Control Unit (ACU) for reading the FluoroBlok^TM tumour cell invasion system gives results comparable to the traditional invasion assay. Compared with traditional invasion systems involving washing, staining and drying steps the introduced method is fast, saving time and labour.

BMG LABTECH readers are able to take multiple readings overtime at a user defined temperature and the Gas Vent or ACU enables an environment conducive to cell proliferation and migration.

FluoroBlok is a trademark of BD Falcon.