A HaemAtlas: characterizing gene expression in differentiated human blood cells
Blood Watkins et al.
113: e1
Supplementary materials for: Watkins et al
Cell purification and purity assessment
Whole blood units (~450 ml) from seven healthy volunteer donors of the Cambridge BioResource at NHS Blood and Transplant (NHSBT) were obtained with informed consent. Blood was taken by venepuncture into a bag containing ACD anticoagulant according to the NHSBT procedures. The blood was allowed to cool on a 4°C surface for 20 minutes prior to the addition of 1 µg/ml of prostaglandin A2 to inhibit platelet activation.
For granulocyte isolation, red blood cells (RBCs) were first removed from the whole blood using gravity sedimentation through HetaSep™ starch solution (StemCell Technologies) following manufacturers’ instructions. Briefly, 3 × 40 ml aliquots of whole blood were each treated with 8 ml of HetaSep™ solution, mixed by inversion and centrifuged at 50 × g for 5 minutes with no brake then allowed to settle for a further 30 minutes at room temperature. The top layer, containing the Nucleated Cell-rich fraction, was transferred to a clean tube and made up to 50 ml with RoboSep buffer (StemCell Technologies) and centrifuged at 800 × g for 10 minutes. The pellet was resuspended in one tenth of the starting volume in RoboSep buffer prior to magnetic separation using the CD66 positive selection kit (StemCell Technologies).
CD4+ Th cells were isolated from peripheral blood mononuclear cells (PBMCs) where the whole blood had previously been treated with Human Monocyte Depletion cocktail RosetteSep™ (StemCell Technologies) following manufacturers’ instructions. Briefly, 40 ml of whole blood was treated with 2 ml of RosetteSep and incubated at room temperature for 20 minutes. The sample was then diluted with an equal volume of phosphate buffered saline (PBS)+ 2% bovine serum albumin (BSA) prior to PBMC preparation.
CD8+ Tc cells, CD14+ monocytes, CD19+ B cells and CD56+ natural killer (NK) cells were isolated from the remaining blood volume. Blood was aliquoted into 50 ml tubes and centrifuged at 150 × g for 20 minutes at room temperature to remove the platelet rich plasma (PRP). The PRP depleted blood was reconstituted to the original starting volume of blood with PBS containing 1.25% BSA and 2 mM EDTA (PBS/BSA/EDTA). PBMCs were then isolated by layering 30 ml onto 15 ml Histopaque-1077 in an Accuspin tube (Sigma) followed by centrifugation at 800 × g for 15 minutes at room temperature with the brake off. The remaining steps were performed at 4°C. PBMC layers were collected and made up to 50 ml with PBS/BSA/EDTA and centrifuged at 1400 × g for 10 minutes. The cell pellets were resuspended in 5 ml PBS/BSA/EDTA, diluted 1 in 5 and centrifuged at 500 × g for a further 10 minutes. A cell count was performed prior to a final centrifugation at 800 × g for 10 minutes. The pellet was then resuspended in RoboSep buffer at a final concentration of 1 × 108 cells/ml prior to magnetic separation. The cells were all isolated using positive selection kits using the markers mentioned above (StemCell Technologies).
Detection of CD248 expression on human T lymphocytes
Blood leucocyte preparation
Leucocyte enriched preparations of buffy coat cells from healthy donors were obtained from the Birmingham Blood Transfusion Service. These preparations had been depleted of platelets and contained sodium citrate as anti-coagulant. Buffy coat was diluted 1:1 with PBS containing 20 Units per ml of Heparin (CP Pharmaceuticals, Wrexham, UK) and layered onto a Ficoll-Paque density gradient (Pharmacia-Biotech, Uppsala, Sweden), at a cell suspension to density gradient volume ratio of ~2:1. The preparation was centrifuged at 450g for 25′ at rt. Leucocytes were harvested into washing buffer (RPMI 1640 bicarbonate buffered medium (RPMI) (Gibco 21875) 2% (v/v) FCS) and washed at 400g for 7′ then 300g for 5′ and resuspended in RPMI 10% FCS.
Tonsil white cell preparation
Normal human palatine tonsils, collected immediately after surgical removal, were placed into petri dishes (Appleton Woods) containing washing buffer. Excess fibrous tissue was removed and samples teased apart to obtain a suspension of cells which was depleted of red cells to provide a leucocyte-enriched preparation, as described for blood leucocytes.
Surface staining
All antibodies were tested at least three times against a given cell target. Results are based on recording the proportion of target cells that had fluorescence intensity above 5% for that target as defined with a negative control antibody. Samples were analysed using a Calibur (Becton Dickinson, Oxford, UK) fluorescence activated cell analyser (FACs).
Two hundred µl of pre washed blood or tonsil samples containing 0.25 × 106 cells were aliquoted into multi-well plates. Cells were spun at 300g for 3′. Supernatants were aspirated and 25µl of supernate from the CD248 clones was added and well contents were mixed. Plates were covered and placed on ice on a shaker for 30′, washed twice with 150µl diluent (PBS 10% FCS), centrifuged at 300g for 3′ and 25µl of appropriately diluted goat anti-mouse fluorescein (Caltag, Burlingame, CA, USA) added to each well and contents gently mixed. Plates were incubated and washed as described above. Twenty-five µl of 10% normal mouse serum (The Binding Site, Birmingham, UK) in diluent was added for 15′ on ice as a blocking stage, the plates were spun and supernatant removed as before.
To identify lymphocyte subsets, cells were stained with 25µl of directly conjugated anti-human reagents as given in the table below combining CD3 with CD4 and CD8 or CD3 with CD45RO and CD45RA. Cells were incubated for 30′ on ice on a shaker then washed as above. 100µl of diluent was added to each well and contents transferred to a 75mm FACs tube (Alpha Laboratories, Eastleigh, UK); 500µl of 1% (w/v) paraformaldehyde (BDH, Poole, England) in PBS pH 7.4 was added. Samples were read on the analyser straight away or stored at +4°C then analysed within 24 hours. Cells were selected by gating on forward (size) and side (granularity) scatter. A minimum of 5,000 events within this gate was analysed.
Bioinformatic analysis
Biological processes
We excluded those PANTHER categories containing ≤20 genes in the human genome, as well as genes annotated as Biological process unclassified. In total, 26 functional categories remained for the subsequent analysis and the expressed genes (n = 11 031) were mapped to these functional categories. The number of genes observed as associated with a particular biological process was calculated for each cell-type. A Z-score was calculated for each biological process using the formula Z = (x − µ)/σ where x is the observed number of genes associated with a particular biological process in a given cell-type, µ and σ are the mean expected number of genes under simulated conditions and corresponding standard deviation, respectively. µ was determined by shuffling the genes 104 times and calculating the number of transcripts associated with a specific biological process under simulation. A highly positive Z-score indicates over-representation of a biological process, while a highly negative score indicates under-representation. When the Z-score is greater (or less) than zero, we computed the p-value of over-representation (or under-representation) associated with the Z-score as the proportion of times during simulation the number of transcripts associated with that biological process was higher (or lower) than the observed number. A p-value cut-off of 0.005 was considered as significant.
The Bloodomics consortium
Academic Medical Center
Amsterdam, The Netherlands
Kastelein J, Trip M
Associazione per lo Studio della Trombosi in Cardiologia
Parma, Italy
Ardissino D, Mannucci P
Domantis Limited
Cambridge, United Kingdom
Steward M, Tomlinson I
European Cardiovascular Genetics Foundation
Cambridge, United Kingdom
Ouwehand W, Bentley D, Barnsley J, Luzzatto L, Vermylen J
Katholieke Universiteit Leuven
Kortrijk, Belgium
Deckmyn H, van Geet C
Luric Datenbank GbR
Eppelheim, Germany
Maerz W, Winkelmann B
Biostatistics Unit, Medical Research Council
Cambridge, United Kingdom
Thompson S, Dudbridge F
Sanquin Blood Supply Foundation
Amsterdam, The Netherlands
van der Schoot CE, Zwaginga JJ
Trium Analysis Online GmbH
Munich, Germany
Daumer M, Scholz M
University of Cambridge
Cambridge, United Kingdom
Farndale R, Ouwehand WH
University College Dublin
Dublin, Ireland
Fitzgerald D, Maguire, PB
University of Leicester
Leicester, United Kingdom
Goodall A, Samani N
Universitair Medisch Centrum Utrecht
Utrecht, The Netherlands
Akkerman JW, de Groot P
Wellcome Trust Sanger Institute
Hinxton, Cambridge, United Kingdom
Deloukas P, Ouwehand WH
