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A case-control study was conducted in Gambian children aged 0-16yrs with severe or uncomplicated as described in the Material and methods section on pg 10 of.[Walther M et al 2009] under subject recruitment, study design and study procedures with the following changes. Uncomplicated and severe case patients were enrolled between September 2008 and January 2009 without the additional control group. In total 117 severe and 156 uncomplicated cases were enrolled.
Blood processing and cell preparation
Blood collected into the heparinised vacutainer tubes (BD) were processed within 2 hours of collection. The blood was centrifuged at 1500 rpm for 5 min at RT. The plasma was then removed, stored at -80°c and replaced by an equal volume of RPMI 1640 (Sigma-Aldrich). An aliquot of the blood suspension was taken to be stained immediately for flow cytometry ex-vivo. The remaining blood suspension then under went density centrifugation over a 1.077 Nycoprep (Nycomed,Sweden) gradient (2500 rpm, 30 min) and the PBMCs were then isolated, washed twice and re-suspended in 5mls of RPMI 1640. The available PBMC in the 5mls was calculated by microscopy (20X objective) using a Neubauer counting chamber and leucocyte counting solution. The PBMC isolate was then stored in liquid nitrogen for later analysis.
The following surface staining cocktail was prepared less than 45 minutes before the samples arrived: 5 µl each of PE anti-CD16b (BD), Per CP anti-CD14 (BD), PE anti-Cy7-CD4 (BD), APC anti-CD19 (BD), Pacific blue anti-CD3 (Ebio) and 4ul of APC-AF 750 anti-HLA-DR (Ebio). An isotype control cocktail was also prepared at the same time containing 2ul of: FITC anti- Mouse IgG1 (BD), PE anti-Mouse IgG2a (BD), Per CP anti-Mouse IgG2b (BD), PE-Cy7 anti-Mouse IgG1 (BD), APC anti-Mouse IgG1 (BD), Pacific blue anti-Mouse IgG2a (Ebio) and APC-AF 750 anti- Mouse IgG2b (Ebio). These cocktails were then vortexed and spun down and placed in the Dark until samples were ready to be stained. 10 Facs tubes were labeled with subject's ID plus 1 of the following: Unstained, Stained, Isotype Control, Comp FITC, Comp PE, Comp Per CP, Comp PE Cy7, Comp APC, Comp Pacific Blue and Comp APC-AF 750. Fresh whole blood suspension was added to the 10 Facs tubes ( manufacturer?) in the following proportions: 20 µl was added in each of the Unstained and Isotype control tubes, 60 µl was added to Stained tube and to the remaining 7 Compensation tubes 10 µl was added to each. Then the surface staining cocktail (29 µl) was added to the Stained tube and the Isotype control cocktail (14 µl) was added to the Isotype control tube. Then 3 µl of the following antibodies were added to the appropriate Compensation tubes: FITC-CD4? (abcam), PE-CD4 (BD), Per CP-CD4 (BD), PE-Cy7-CD4 (BD), APC-CD4 (BD), Pacific blue-CD3 (Ebio) and 4ul of APC-AF 750-CD8 (Ebio). Facs tubes were vortexed and incubated for 35 min at 4° in the dark. After the incubation for surface staining was over, 4 volumes of the diluted FACS lysing buffer (Ebioscience?) was added to the Facs tubes then vortexed and incubated at RT in the dark for 5 min. Then immediately afterwards the tubes were filled till the top with FACS buffer (1x PBS, 0.1% sodium azide (Sigma-Aldrich), 2mM EDTA and 1% FPS (Invitrogen Life Technologies)) and quickly spun at (1400rpm, 5min). The supernatants were then taken off, and the pellets re-suspended. Then another 3mls of FACS buffer was added per tube and washing repeated by centrifuging at 1400 rpm for 5 min and decanting the supernatants. A 100µl of Cytofix/Perm buffer (BD) was then added to all samples, and pulse vortexed and then incubated at 4° C for 20 min in the dark. After incubation the samples were washed twice with 1 ml of permwash buffer (BD) (diluted 1:10 with dH20).
Then 5% of mouse serum was added to the Facs tube labelled 'stained' and incubated in the dark at 4° C for 15 min. Then immediately after without washing 3.5µl of FITC anti-HO-1 (abcam) was added in the stained tube and left to incubate at 4° for 30min in the dark. Then after incubation the Stained labelled tube was washed twice in 1ml of permwash buffer (BD). All tubes were then re-suspended in a drop of Facs buffer and acquired on a 3 laser/9 channel CyAnTM ADP flowcytometer using Summit 4.3 software (Dako). Analysis was performed using FlowJo (Tree Star Inc.).
Cytoplamic and Nuclear extraction
Performed on separate PBMC and Granulocyte cell suspensions of the same sample. As directed by the Manufacturer's guide lines for cell suspensions in the Kit (kit manufacturer?)
HO-1 Detection in the Plasma.
This procedure was performed as direct by the manufacturer's guide lines in the HO-1 (human) ImmunoSet Kit # 960-800 using the plasma samples aliquoted and frozen during the blood processing and cell preparation.
Heat induction assay.
Blood samples (D28) collected into the heparinised vacutainer tubes (BD) were spun down at 1700 rpm for 5 min at 4°C. The plasma was then removed, stored at -80°c and replaced by an equal volume of RPMI 1640 (Sigma-Aldrich) and any evidence of haemolysis was noted. Then 200 µl of a blood sample was added to each of the following 5 labelled RNase free Eppendorf tubes (manufacturer?): IM (Immediate), RT (Room temp), 37.5 (37.5 degrees), 37.5H (37.5 degrees + Hemin), 40 (40 degrees s). To Eppendorf 37.5H and 40H 2 ul of hemin (1mM) (Sigma Aldrich?) was added. Then Eppendorf tubes labelled 40 and 40H were placed in a 40°C water bath for 3 hours. The 37.5 and 37.5H labelled tubes were placed in the incubator set at 37.5°C for 3 hours. RT labelled Eppendorf tube was left in the hood for 3 hrs. The tube that was labelled IM, was then diluted 1:1 with RNase free water (manufacturer?) making a 400 ul cell suspension. Then 1.2 ml of Trizol LS reagent(manufacturer?) was added to the tube. The solution was then pipetted up and down thoroughly an vortexed for at least 30s, then stored in a freezer at -60°C. After 3 hours of incubation this same procedure was then performed on the remaining tubes.
This was performed as described in the Material and methods section on pg 11 of.[Walther M et al 2009] under RT-PCR. A 1ml aliquot of each blood sample was stored at -80°C after being collected into a tube containing RNA stabilizing agents (PAXgeneTM Blood RNA System, Per-AnalytiX)). RNA was isolated from these samples along with samples stored in Trizol ls reagent from the HI assay following the manufacturer's instructions (Pre-AnalitiX). cDNA was then synthesised using TaqMans Reagents for Reverse Transcription (Applied Biosystems) following the manufacturer's instructions. RT-PCR was performed using the TaqMans Probe PCR kit. Primers used were: HO-1 (details for this pimer or reference?) Ribosomal 18s RNA was used as the reference house-keeping gene (TaqMans Ribosomal RNA Control Reagents, Applied Biosystems). Samples were run in duplicates on a DNAEngine Opticons (MJ Research), and analysed using OpticonMonitor 3TM analysis software (BioRad).
Plasma sample aliquots stored at -80°c were thawed and centrifuged (5 min 4°c, 1,100g) to remove contaminants and then passed it through a Microcon YM-3 column (Millipore)(60 min at 14°C, 21,000g) to remove proteins (MW >3 kDa). Free heme was quantified in the protein-depleted plasma using a chromogenic assay according to the manufacturer's instructions (QuantiChrom heme assay kit,
Facs tube manufacturer
RNase Eppendorf tubes manufacturer
Facs lysis buffer manufacture Ebio?
Nuclear and cyoplasmic extraction kit?
Blood processing. Some complete medium (manufacturers), incomplete medium and Nycoprep were warmed up to RT. Then the Nycoprep was layered in a falcon tube and then the blood was layered carefully in a 1:1 ratio on top of the Nycoprep. The falcon tube was then centrifuged at 2500 RPM for 25 minutes with no brake. After centrifugation a distinct band can be seen near the top of the tube and the RBC formed a pellet at the bottom with Nycoprep on top. In between these two a cloudy liquid fraction that sometimes contained an additional band was seen. Using a sterile Pasteur pipette the cloudy liquid fraction consisting of Peripheral Blood Mononuclear Cells was collected and placed in a 15ml falcon tube. The tube was then toped off to 14 ml with incomplete RPMI and centrifuged at 1700 rpm for 5 min and the supernatant then discarded. This step was repeated twice and then the PBMC pellet was re-suspended in 5mls of complete medium and a 10 ul aliquot placed in an Eppendorf tube and the remaining PBMC solution spun down as before. The Neubauer counting chamber was prepared. Then 90 ul of the leucocyte counting solution was added to the eppendorf containing the 10 ul of cell suspension Mix and left for 30 seconds. Then 20 ul of this mix was taken with a pipette and one of the grids of the chamber was filled by putting the tip of the pipette at an angle of 45 degrees. Then using the microscope with a 20X objective with the condenser iris closed sufficiently to give good contrast, the rulings of the chamber and white cells were focused. Then the number white cells in two of the large corner squares of the chamber marked w1 and w4 were counted and the amount of available PBMC in the 5mls was calculated as follows
Cells counted x 2(to give total for 4 squares) x 50,000(to adjust for volume in 5ml) / 0.4 (chamber volume) = Cells in 5ml