Jurkat cell lines
4.2 MA TERIALS AND METHODS
The experimental work presented in this chapter was carried out at Nottingham Trent University (NTU), Faculty of Health Sciences, United Kingdom (UK), with supervision and guidance from Mrs Anne Cox, Dr Claire Scholfield and Professor Martin Griffin (July-September 1999) in collaboration with LumiTech™ at the Nottingham City Hospital.
Financial support was kindly provided by the National Research Foundation (NRF), SA.
Only the effects of FB I and FA were assessed in this international collaborative study, the reason being that this work was part of the initial experiments carried out and the other mycotoxins included in the assessments in Chapters 3, 5, and 6 were added in later in the time line of work presented in this dissertation.
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4.2.1 Materials
Cell culture materials, media, antibiotics and sterile disposable consumables were purchased from Sigma Chemical Company (UK). Pre-sterilised Corning tissue culture flasks (75cm2) were used as culture vessels, sterile clear 96-well microtitre plates (CoStar) and 96 well white-walled clear-bottomed luminometer plates (Wallac) were used for the assays. Aseptic protocols were adhered to. The N2a cell line was obtained from the cell culture facility at NTU, and the Jurkat cell line from the cell culture facility at Leicester University, UK. A Lucy 1 Anthos Luminoskan Labsystems microplate luminometer equipped with reagent dispensers capable of injecting 20I-.t1 volumes was used, enabling full automation of the assay.
The Jurkat cell line was cultured in RPMI-1640 cell culture media containing 10% FCS, 2mM L-glutamine, 2% Penstrep and 2mg.mri gentamicin sulphate. The N2a cell line was cultured in DMEM supplemented with 10% FCS (v/v), 2mM L-glutamine, 2mM sodium pyruvate and 2% (v/v) penicillin streptomycin.
4.2.2 Methods
4.2.2.1 The ApoGlowTM assay
ATP contamination can be a source of error for the ApoGlowTM assay. The surface of the skin contains significant levels of ATP, therefore gloves were worn when performing the assay, and ATP-free microtitre plates and tubes were also used to minimize errors.
The N2a cells were cultured in 75cm2 culture flasks until confluent, trypsinised, and following a cell count, 2.5 x 104 cells were dispensed into each well of the microtitre plates. The Jurkat cell line, being a suspension cell line, did not require trypsinisation. To minimize dilution of the mycotoxins by the media in the pure cell suspension, cells were concentrated in small volumes of CCM (~1 0-15 1-11).
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4.2.2.2 Preparation of the mycotoxin stock solutions
Fumonisin BI and FA (lmg) were dissolved in 30~1 DMSO and 970~1 CCM (using the defined media required for each cell line), to a stock solution of 1 mg.mrl
. The control solution for each toxin was made up of30~1 DMSO and 970~1 CCM. A range of dilutions of both the mycotoxin stocks and controls were assessed as shown in Figures 4.2- 4.3. In addition, the cells were exposed to equal volumes ofa combined solution of FBI and FA at each concentration. For example, at 100~g.mrl (FBI+FA), if 1O~1 of cell suspension was used to obtain 2.5 x 104 cells per well, this volume was topped up to a total of 100~1 per well with 45~1 of FBI and 45~1 of FA using separate stock solutions of 100~g.mrl of each mycotoxin. Duplicate testing of each mycotoxin was carried out on both cell lines. The final volume of cell suspension and diluted mycotoxins together was 100~1 according to the manufacturers' instructions. The culture plates were then incubated at 37°C for between 22.5-24 hours, after which the ApoGlowTM assay was carried out.
4.2.2.3 Outline of the method
Before cellular adenylate nucleotides were measured by bioluminescence, they were first extracted from the cell. For the N2a adherent cells, 100J .. tl of nucleotide releasing reagent (NRR) was added to each well of the microtitre plate using a multi-channel pipette, and extraction was allowed to occur for five minutes at R T. The extracts were transferred to an opaque white microtitre plate and loaded into the luminometer. Where the cells were cultured in white walled clear-bottomed tissue culture plates, extraction and measurement of adenylate nucleotides was carried out in the same plate. The assay procedure differed slightly between the N2a adherent and the Jurkat non-adherent cells. With the Jurkat non-adherent cells, one bottle of nucleotide monitoring reagent (NMR) (20m I) was reconstituted in one bottle of NRR (20ml), enabling simultaneous release and measurement.
4.2.2.4 Equipment
The assay was fully automated usmg the Lucy 1 Anthos Luminoskan Labsystems microplate luminometer. The data was expressed as relative light units (RLUs).
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Dispenser 1 was primed with NMR and set to dispense 20!!1 in the case of the N2a adherent cells, and 100!!1 for the Jurkat non-adherent cells. The instrument was programmed to dispense into each well and take an immediate one-second integrated reading before moving on to the next well. Dispenser 2 was primed with ADP converting reagent, and also programmed to dispense 20!!1 into selected wells and take an immediate one-second integrated reading before moving on to the next well. At all times the luminometer dispensing lines were kept scrupulously clean. Any residual luciferase in the dispensing line was removed using a cleaning reagent that neutralised the enzyme.
The specifically formulated luminescence cocktail (NMR) was automatically added to the wells and an ATP reading (A) was taken. After 10 minutes, the second injector added the ADP Converting Reagent and a second reading was taken (B; baseline ADP levels). After a further 5 minutes, the final converted ADP reading was taken (C). These three values were then used to calculate the ADP:ATP ratio.
4.2.2.5 Interpretation of results
The ADP:ATP ratio was then calculated from measurements A, Band C as follows:- (C-B)/A
Interpretation of different ratios obtained within each experimental situation varied according to the cell type and conditions used. However, the following criteria, provided by the manufacturers of the assay were used as guidelines: -
1. If a test gives markedly elevated ATP values (compared to seeding cell densities) with no significant increase in ADP levels, this indicates proliferation. Stimulation with growth/mitogenic factors will induce proliferation, and thus lead to an increase in ATP readings.
2. If a test gives considerably lower ATP levels than control but greatly increased ADP, this indicates necrosis.
3. If a test gives similar or slightly higher levels of ATP, and with little or no change in ADP compared to control, this indicates that the treatment arrests proliferation but does not kill cells.
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4. If a test gives lower levels of ATP to control but shows an increase in ADP, this indicates apoptosis. These ratios vary according to the degree of apoptosis in the cell population.
According to the manufacturers guidelines and the LumiTech ™ representative (Scholfield pers comm., 2002), as a further guide on interpretation of data, ratios greater than twice the control values, but less than four times control values, were an indication that cells were undergoing apoptosis. Ratios four times greater than the control values were an indication of necrotic cell death. Ratios slightly lower than the ADP: ATP ratio of control cells were indicative of cells undergoing growth arrest, and those with ratios significantly lower, as an indication of cell proliferation.
The experiments presented were performed in duplicate and the results are presented as means ± SD.