Protein detergent fractionation
Cytosolic and membrane proteins from the collected subsets of NK cells were obtained
by detergent fractionation  Pellets of CD56bright (2x106 cells/pellet), CD56dim (107
cells/pellet) and NKT (5x 106 cells/pellet) cells were suspended in 100 :L ice-cold Digitonin
Buffer (10 mM Piperazine-N,N'-bis2-ethanesulfonic acid (PIPES buffer), pH 6.8, 0.015% (w/v)
digitonin, 300 mM sucrose, 100 mM NaCl, 3 mM MgCl2, 5 mM EDTA, 1 mM
phenylmethylsulphonylflouride (PMSF)). Cells were permeabilized by gentle agitation on ice
for 10 min, then centrifuged (10 min, 500 xg) and the supernatants (cytosolic proteins) were
stored at –80oC. The remaining pellet was re-suspended in 100 μL of ice-cold Triton X-100
extraction buffer (10 mM PIPES pH 7.4, 0.5% (v/v) Triton X-100, 300 mM Sucrose, 100 mM
NaCl, 3 mM MgCl2, 5 mM EDTA, 1 mM PMSF) and further agitated on ice for 30 min, then
centrifuged (10 min, 500 xg). The supernatants (membrane-bound proteins) were removed and
stored at –80oC.
A Protein Clean-Up Kit (Amersham Biosciences, Pittsburgh, PA, USA) was used to
precipitate membrane and cytosolic proteins while removing detergents, salts, lipids and nucleic
acids according to manufacturer’s recommendations. Briefly, the samples were mixed with 300
μL of Precipitant, incubated on ice for 15 min, centrifuged (12000xg, 5 min) and supernatants
were discarded. Forty μL of Co-Precipitant were layered onto the pellets, incubated on ice 5
min, centrifuged (12000xg, 2 min), and pellets were re-suspended in 25 μL of distilled water.
After washing with pre-chilled (-20ΕC) Wash Buffer, samples were vortexed to disperse then
incubated on ice for 30 min. After collection by centrifugation (12000xg, 5 min), the protein
pellets were air dried, then re-suspended in sterile water.
Cytosolic and membrane protein samples were quantified in triplicate using the
bicinchoninic acid (BCA) protein assay (Pierce, Rockford IL, USA) in which samples diluted in
200 μL of BCA Assay Working Reagent were incubated for 30 min at 37oC, then assessed for
absorbance at 560 and 620 nm (Thermo LabSystems Multiskan Ascent Plate Reader).
Depending upon availability, a minimum of 5 :g to a maximum of 10 :g of each protein
sample were diluted in 1X SDS gel-loading buffer (50mM Tris-HCl (pH 6.8), 2% β-
Mercaptoethanol, 0.1% Bromophenol blue, and 10% glycerol). Heated samples of membrane
and cytosolic proteins were loaded into alternate lanes of 12% polyacrylamide gels (Tris HCl
(1.5 M, pH 8.8), Tris HCl (0.5 M, pH 6.8), Acryl/Bis (40%, 37:5:1), SDS (10%), Ammonium
Persulfate (10%), TEMED (0.01%), and MilliQ water)(Bio-Rad, Hercules CA, USA).
Prestained SDS-PAGE Standard Broad Range ladder (Bio-Rad) was loaded in the first and last
lanes to confirm transfer and Magic Mark Western Blotting Protein Standard (Invitrogen) was
used for determination of molecular weight after chemiluminescence. After electrophoreses at
100 V, proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad) at
350 amperes for 3.5h at 4oC. The membranes were rinsed and equilibrated in 1X tris buffered
saline (TBS; 0.5M Tris base, 9% NaCl, pH 7.6) and blocked 2h in 3% carnation milk in TBS-
Tween 20 (0.1%). The blots were then cut in horizontal strips for large, medium and small
protein detection and labelled overnight at 4oC with primary antibodies (in TBS-Tween 20
(0.1%), 1% milk) at optimized dilutions (Table I). Membrane strips were washed, then
incubated 1 h with HRP-conjugated secondary antibodies (in TBS-Tween 20 (0.1%), 1% milk).
which were detected by 5 min incubation in SuperSignal® West Femto Maximum Sensitivity
Substrate (SuperSignal West-Femto, Pierce, Rockford IL, USA) and exposure to Kodak
Scientific Imaging Film (Eastman Kodak Company). Images were taken using Alpha Innotech
FluorChem™ 8000 Imaging System (Alpha Innotech, San Leandro CA, USA) for densitometry
analysis with FluorChem™ Imaging Software (Alpha Innotech). Spot volume and density within
each band was measured then each result was normalized to its cytosolic or membrane control
GAPDH or CD45, by dividing the integrated density volume (IDV) obtained by the Alpha
Innotech FluorChem program by the IDV of either CD45 or GAPDH to account for differences
in loaded amounts.
After chemiluminescent detection, membrane strips were rinsed thrice in 1X TBS-
Tween 20 (0.1%) for 10 min each. Blots were stripped with Restore Western Blot Stripping
Buffer (Pierce) for 20 min at room temperature, washed in TBS 3 times for 10 min, assessed for
chemiluminescent signal by exposure to a fresh piece of film, and then re-probed with another
size appropriate antibody. Blots were stripped no more than three times.
Extraction of mRNA and RT-PCR
The μMACS mRNA Isolation Kit was used to extract mRNA from fractionated subsets
of human CD56bright and CD56dim subsets according to manufacturer’s instructions. Briefly,
pellets not exceeding 107 cells, were lysed with 1 mL Lysis Binding Buffer, vortexed 3 min,
then centrifuged (3 min, 4000xg). Lysates were applied to Lysate Clear Columns and
centrifuged (3 min, 13000xg). Then 50 μL of Oligo-μdT Beads were added to the cleared lysate
and applied to prepared MACS Type μ columns in a μMACS separator magnet. Magnetically
labelled mRNA was retained as the column was rinsed twice with Lysis Binding Buffer and 4
times with Wash Buffer, then eluted with 120 μL of Elution Buffer at 68oC. RNA was
quantified by spectrophotometry at 260nm (BioPhotometer, Eppendorf, Westbury NY, USA),
and its integrity assessed by electrophoreses on a 1% agarose gel.
mRNA was reverse transcribed by adding 2 μL of Oligo (dT)12-18 (Invitrogen,
Burlington, ON) to 2μL mRNA, incubating 10 min at 70oC, then chilling to 4oC. A mix of 4 μL
5X First Strand Buffer, 1 μL 10mM dNTP, 2 μL 0.1M Dithiothreitol and 0.5 μL RNaseIN
(Invitrogen) were added, held 2 min at 42oC before adding 1 μL of SuperScript II RT
(Invitrogen) for incubation for 60 min at 43oC, 15 min at 70oC, then 10 min at 4oC. Finally, 1 μl
of SuperScript II RNase H–Reverse Transcriptase (Invitrogen) was added for 20 min at 37oC.
Conventional PCR was used to amplify transcripts of interest as summarized in Table II.
Primers were designed using Primer 3 Software (Whitehead Institute for Biomedical Research,
Cambridge MA, USA). A mix of 2 μL 10x PCR Buffer (22 mM Tris-HCl, pH 8.4, 500 mM
KCl) (Invitrogen), 0.4 μL 10 mM dNTP (Invitrogen), 0.3 μL RedTaq DNA polymerase (Sigma-
Aldrich), 1 μL each of 10 μM forward and reverse primers, 13.3 μL distilled water and 2 μL of
template cDNA together in a PCR thin walled 0.5 mL tube. Amplifications started with a 3 min
template denaturation step at 94oC, followed by 35 cycles of denaturation for 20s at 94oC and a
combined primer annealing/extension at gene specific primer temperatures for 30s. All samples
were amplified in triplicate and compared to the housekeeping gene glyceraldehyde-3-
phosphate dehydrogenase (GAPDH). Distilled water, in place of template cDNA, was used as a
negative control in all reactions. For analysis of reproductive hormone receptors on NK cell
subsets, human ovary and placenta cDNA were used as controls (US Biologicals, Swampscott,
PCR products were electrophoresed on a 1% agarose gel, bands excised, and cDNA was
purified using QIAquick Gel Extraction Kit (Qiagen, Mississauga ON, Canada). The resultant
PCR-generated cDNA was quantified by spectrophotometry and product identities were
confirmed by sequencing (Sequencing Facility, Robarts Research Institute, London, Canada).