CHAPTER 3 MATERIALS AND METHODS
3.5 Lactate Dehydrogenase Detection
3.5.1 Principle
The CytoTox96® Non-Radioactive Cytotoxicity Assay (Promega) was used to quantify the level of chlamydia-induced cytotoxicity in HaCaT and ME-180 cells. This assay quantifies the level of lactate dehydrogenase (LDH), a cytosolic enzyme which is released upon cell lysis (Sepp et al 1996). The level of LDH released into the culture supernatent is detected using a coupled enzymatic reaction in which the tetrazolium salt is converted to a
red formazan product. The wavelength absorbance data recorded are used to calculate the percentage cytotoxicity.
Since there are several factors which affect the absorbance at the wavelength used in this assay, including FBS and phenol red which were both present in the chlamydia growth medium for experiments (CGM-E), four controls were set up:
• The host cell spontaneous LDH release utilizes the supernatant from uninfected cells and corrects for the spontaneous release of LDH from the host cells.
• The host cell maximum LDH release utilizes the supernatant from uninfected cells which have been lysed. It is required in calculations in order to determine 100% LDH release.
• The volume correction control comprises culture medium without cells to which the lysis solution has been added in order to compensate for the volume change caused by the addition of lysis solution to the host cell maximum LDH release control.
• A culture medium background control comprising only cell culture medium without cells corrects for the LDH activity contributed by serum in the cell culture medium, as well as the presence of phenol red.
3.5.2 Methodology
HaCaT or ME-180 cells were seeded (1.5 x 104 cells in 100µl CCM per well) into flat- bottom 96-well TC microtitre plates and incubated at 37°C overnight to yield an 80%
confluent monolayer the following day. Three wells for each cell line were trypsinized and the cell count used to calculate an appropriate dilution of the stock chlamydial inoculum to
produce a MOI of 0.025. This low MOI ensured that only a few cells became infected in the first instance, allowing sufficient uninfected neighbouring cells for the progeny EB to infect after completion of the chlamydial lifecyle.
CCM was aspirated from the wells and replaced with 100µl CGM-E comprised of RPMI- 1640 (BioWhittakerTM) with 10mM HEPES, 2mM L-glutamine, gentamicin (10µg/ml), Amphotericin B (5µg/ml), glucose (5.4mg/ml) and 10% FBS. Cycloheximide was not used in any experiments, because it inhibits eukaryotic cell replication by interfering with the 60S ribosomal subunit (Elela and Nazar, 1997), which would prevent eukaryotic host cells from behaving as they would in an in vivo situation.
Cells were infected in triplicate with 3 C. trachomatis reference strains (serovars L1, L2 and L3), 1 OG strain (serovar E), 3 LGV clinical isolates or 50µl sterile SPG for the negative control and maximum LDH release control. After addition of the inoculum or SPG, plates were centrifuged at 1200 x g for 1 h, incubated at 37°C or 33°C for 1 h, then the inoculum removed and replaced with 150µl fresh CGM-E. At this point 150µl CGM-E was added to 6 empty wells for subsequent use as the volume correction control and culture medium background control. Plates were incubated at 37°C or 33°C for 5 days.
Each day post-infection, 1 plate from each cell line and both temperatures for the HaCaT cell line, was removed from the incubator for use in the LDH assay and chlamydial growth curve study. The remaining plates were centrifuged at 1200 x g for 1 h each day then returned to the incubator. On the second day post-infection 100 µl fresh CGM-E was added to all remaining wells, while 50 µl fresh CGM-E was added on day 4.
For the LDH assay, lysis solution was added to the volume correction and maximum LDH release control. The volume added was 10% of the volume of CGM-E in the well, therefore on days 1 and 2, 15µl was added, on days 3 and 4, 25µl lysis solution was added, while 30µl lysis solution was added on day 5. The solution in each well was mixed by stirring with a pipette tip, and the plates returned to the incubator for 45 minutes until the cells had lysed. The solution was mixed by pipetting up and down, then cellular debris pelleted by centrifugation at 250 x g for 4 minutes.
Twenty-five microlitres supernant was transferred from each well to a clean well on a new flat-bottom 96-well TC plate. Because of the high LDH activity, the superant was diluted with 25µl PBS to produce a final volume of 50µl. Reconstituted substrate mix (50 µl) was added to each well. The plates were covered with foil and incubated at room temperature for 20 minutes. The reaction was stopped with 50µl stop solution (1M acetic acid) and the absorbance read at λ 492nm using an Anthos 2010 version 1.7 microplate reader.
Before calculation of the percentage cytotoxicity, the average absorbance of the volume correction control was subtracted from the maximum LDH release control, and the average absorbance values of the culture medium background control subtracted from all other absorbance values.
The corrected absorbance values were used to calculate the percent cytotoxicity in the following calculation:
% cytotoxicity = Experimental – Host Spontaneous x 100 Host Maximum – Host Spontaneous
This experiment was performed 3 times in triplicate.
No effector cell (chlamydia) spontaneous LDH release was measured since C. trachomatis is an obligate intracellular organism, and thus unable to replicate in the absence of a eukaryotic host. These organisms rely on adenosine triphosphate (ATP) from their host, and there are no reports of this organism possessing LDH, an enzyme of the glycolytic pathway.