ANTECEDENTES HISTORICOS

Overview: Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and cultured for 24 hours with and without lipopolysaccharide (LPS) stimulation. Cell conditioned media was collected and frozen for future analysis of PGE₂/PGE₃ content. PBMCs were then either frozen for future COX RNA analysis, or further processed to obtain nuclear extract which was then frozen for subsequent batch analysis of nuclear NFκB activation.

4.3.1 PBMC culture

Blood collected in CPT™ tubes at each study visit was centrifuged within 3 hours of venepuncture at 1500 x g for 20mins to give a layer of mononuclear cells (Figure 4.3). The mononuclear cell layers were aspirated into two falcon tubes, washed with 15mls of Dulbecco’s phosphate buffered saline (DPBS) and centrifuged at 300 x g for 15 mins. The wash step was repeated. The cells from each tube were re-suspended in 6 mls GlutaMAX + 10 % foetal calf serum (FCS) with or without 1µg/ml Escherichia coli serotype 026:B6 LPS (Sigma-Aldrich, MO, USA) respectively. After re-suspension in culture medium, a 20µl sample of LPS-stimulated and LPS unLPS-stimulated cells was taken, diluted 1:10 in DPBS and cells counted using a Neubauer chamber. The remaining cells were transferred to a 6 well culture plate (Figure 4.4) and incubated for 24hrs at 37^oC.

Figure 4.3. Separation of mononuclear cells after centrifugation of CPT™ tubes

Figure 4.4. Layout of 6 well plate for culture of PBMCs. Following 24 hour culture, cells from wells 3 and 6 were harvested and frozen for future COX RNA analysis. Cells from wells 1,2,4 and 5 were processed further to obtain nuclear extract for the measurement of nuclear NFκB activation.

‘+’ i.e. LPS stimulated

‘-‘ i.e. LPS unstimulated

1 2

4 5 6

3

Culture medium from each well was aspirated into four 15µl falcon tubes as described below:

i) Wells 1+2 : LPS stimulated for NFκB nuclear expression ii) Well 3 : LPS stimulated for COX RNA expression

iii) Wells 4+5 : LPS unstimulated for NFκB nuclear expression iv) Well 6 : LPS unstimulated for COX RNA expression

Tubes were centrifuged at 300 x g at 4^oC for 10mins, and the culture medium aliquoted into cryogenic vials and stored at -80^oC until subsequent analysis of culture medium PGE2 and PGE3 content by LC-MS/MS. Cell pellets were left in the respective falcon tubes.

4.3.2 Harvest of PBMCs for COX expression analysis

Wells 3 and 6 were washed with 7mls DPBS, cells gently scraped using a cell scraper, and the cells aspirated back into their respective falcon tubes and centrifuged at 300 x g at 4^oC for 10mins. The supernatant was discarded and cells re-suspended in 1ml DPBS, transferred to a microcentrifuge tube and centrifuged at 300 x g at 4^oC for 5mins. The supernatant was discarded, cells re-suspended in 1ml TRIzol® (Invitrogen, Life Technologies, CA, USA) and stored at -80^oC.

Analysis of COX RNA expression by real-time polymerase chain reaction (RT-PCR) was not performed due to time and funding constraints. Samples remain stored for analysis at a later date.

4.3.3 Isolation of PBMC nuclear extract

PBMC nuclear extract was obtained using the Active Motif Nuclear Extract Kit (Active Motif, CA, USA). Wells 1,2,4 and 5 were washed with 7mls of phosphate buffered saline - phosphatase inhibitor solution (PBS-PI), cells gently scraped using a cell scraper, and aspirated back into their respective falcon tubes. Addition of phosphatase inhibitor protects proteins from dephosphorylation. Tubes were

centrifuged at 300 x g at 4^oC for 10mins, the supernatant discarded, and cells re-suspended in 1ml PBS-PI. Cells were transferred to microcentrifuge tubes and centrifuged at 300g at 4^oC for 5mins. The supernatant was discarded and cells re-suspended in 1ml hypotonic buffer solution and incubated on ice for 15mins.

Hypotonic buffer causes cell swelling, making the cell membrane more fragile.

50μl detergent was added to each microcentrifuge tube and vortexed gently.

Detergent causes leakage of cytoplasmic proteins into the supernatant. Tubes were centrifuged at 14,000 x g at 4^oC for 30secs, and the supernatant (cytoplasmic fraction) aspirated and stored in cryogenic vials at -80^oC.

The nuclear pellet was re-suspended in 100µl of complete lysis buffer, vortexed for 10 seconds, and incubated on ice for 30mins on rocking platform at 150rpm. This causes lysis of nuclei, and nuclear proteins are solubilised in the lysis buffer, which contains a protease inhibitor cocktail to protect proteins from proteolysis. Tubes were centrifuged at 14,000g for 10mins at 4^oC, and the supernatant (containing the solubilised nuclear proteins) aspirated and aliquoted into microcentrifuge tubes and stored at -80^oC until subsequent batch analysis of nuclear NFκB activation (see section 4.3.4).

4.3.4 Quantification of PBMC nuclear NFκB activation

Overview: Nuclear NFκB activation was quantified using the Active Motif TransAM™ NFκB p65 enzyme-linked immunosorbent assay (ELISA) (Active Motif, CA, USA). Each well of the supplied 96-well plate is pre-bound with a double-stranded oligonucleotide containing the NFκB consensus binding site. Activated NFκB in the PBMC nuclear extract samples binds to the oligonucleotide, and in doing so an epitope on the p65 NFκB subunit is exposed. A primary antibody directed against this epitope is added. The primary antibody therefore only binds to activated, DNA-bound NFκB. Binding of an anti-rabbit horseradish peroxidise

(HRP)-conjugated secondary antibody provides a colorimetric reaction which can be quantified by spectrophotometry. Nuclear extract from Jurkat cells (an immortalised T-lymphocyte cell line) was supplied with the assay for use as a positive control.

Protein concentration of samples: The protein concentration in each nuclear extract sample was determined using the Bio-Rad DC Protein Assay (Bio-Rad, CA, USA), a modification of the Lowry assay.(284, 285) Bovine serum albumin (Sigma-Aldrich, MO, USA) was prepared in DPBS to standard concentrations of 2mg/ml, 1mg/ml, 0.5mg/ml and 0.1mg/m. 5µl of each standard and sample were added in duplicate to separate wells of a 96-well plate. 25µl reagent A’ and 200µl reagent B were added to each well and incubated at room temperature for 15 mins. Optical density was read at 750nm on an Opsys MR™ plate reader (Dynex Technologies, VA, USA). The mean of the duplicate samples and standards was calculated. A graph of OD vs. protein concentration was plotted for the standards, and a line of best fit generated. The protein concentration of each sample was calculated according to the equation for the line of best fit.

Binding of NFκB to its consensus sequence: For each nuclear extract sample, 5µg of nuclear extract protein diluted in 20µl of complete lysis buffer (CLB) were added in duplicate to the TransAM™ NFκB 96-well plate. 5µg Jurkat nuclear extract in 20µl CLB and 20µl CLB were added in duplicate as positive and negative controls respectively. The plate was left to incubate for 1hr at room temperature at 100rpm on a rocking platform. Wells were washed three times with 200µl wash buffer.

Binding of primary antibody: 100µl of NFκB antibody (1:1000 dilution in antibody binding buffer) was added to each well and the plate incubated for 1hr at room temperature. Wells were washed three times with 200µl wash buffer.

Binding of secondary antibody: 100µl of HRP-conjugated antibody (1:1000 dilution in antibody binding buffer) was added to each well and the plate incubated for 1hr at room temperature. Wells were washed four times with 200µl wash buffer.

Colorimetric reaction: 100µl developing solution was added to each well and incubated for 4.5 mins at room temperature. 100µl of stop solution was added, and OD measured at 450nm and 630nm read on an Opsys MR™ plate reader (Dynex Technologies, VA, USA).

Quantification of NFκB: Optical density at 630nm (reference OD) was subtracted from OD at 430nm. The mean corrected OD of each duplicate sample was calculated, and divided by the mean corrected OD of the positive control wells to give the nuclear NFκB activation of each sample relative to that of the Jurkat nuclear extract standard.