Purification of recombinant Cyclin B1
Baculovirus infected insect cells expressing recombinant human cyclin B1 was a generous gift from Susan Wormsley (PharMingen, San Diego, CA). To purify cyclin B1, 2.5×108 cells were suspended in 7 ml lysis buffer [50 mM Tris HCl, pH 7.0, 150 mM NaCl, 0.1% Na Azide, 2 mM phenylmethylsufonyl fluoride (PMSF) and 10 µl/ml protease inhibitor cocktail (Sigma P8340)]. An equal volume of lysis buffer with 2% Nonidet P-40 (NP-40) was added, and the lysate was stirred for 1 h at 4°C. Insoluble material was removed by centrifugation at 15,000 g for 15 min at 4°C; 1 ml aliquots of supernatant were frozen in a dry ice/EtOH bath and stored at −80°C.
Anti-cyclin B1 monoclonal antibody (GNS1, PharMingen) was conjugated to Affi-Gel Hz (BioRad, Hercules, CA) as directed. Four mg IgG was oxidized and coupled to 4 ml of Affi-Gel Hz. Absorbance at 280 nm was used to quantify coupling efficiency (44%).
Lysate was added to the affinity column and the column was washed using lysis buffer with 0.5% NP-40 (solution A) then with solution A with 0.5 M NaCl until A280 returned to base line. Cyclin B1 was eluted with 1M sodium propionate, 150 mM NaCl, 10% dioxane, and 0.5% NP40 at pH 5.0 (solution E). A shallow gradient to 50% solution E followed by a steep gradient to 100% solution E was used to collect fractions. Samples were frozen in dry ice/ethanol and stored at −80°C. Fractions were monitored by Western blotting. Cyclin B1 fractions were pooled and concentrated in 30K MW MSI Ultrafuge (Westboro, MA) centrifuge filters. The concentrate was subjected to anion exchange chromatography as follows. Pooled fractions were re-equilibrated in solution A without NP-40 (EconoPac 10 DG, BioRad) then concentrated in MSI Ultrafuge or Millipore (Bedford, MA) Ultrafree-4 centrifuge filters and mixed with a Macro-Prep High Q Support (BioRad) anion exchange resin in solution A. After centrifugation the preparations were concentrated again to less than 1 ml.
Cell Lines and Culture
PC3, DU145, RKO, HeLa, and 22Rv1 were grown in Dulbecco's modified Eagle medium (DMEM) with 5% FBS and 5% calf serum (Sigma, St. Louis, MO). K562 was grown in RPMI 1640 (Gibco, Grand Island, NY) with 10% FBS. All cultures were maintained at 37°C in 5% CO2.
Cell line lysates
Adherent cells harvested with trypsin-EDTA or nonadherent cells were counted with a Coulter Counter (Coulter, Hialeah, FL), and then washed in PBS. Lysis buffer was added to give a final cell concentration of between 5×106/ml and 5×107/ml, depending on the cyclin B1 level in the cell line. Cells were lysed with SDS lysis buffer (1% sodium dodecyl sulfate (SDS), 2% NP-40, 1% Na deoxycholate, 137 mM NaCl, 20 mM Tris HCl, pH 7.5) and DNA was sheared by syringing with a 26-gauge needle. SDS concentration was varied to 5 or 23%. Other extraction buffers used were RIPA (1% Na deoxycholate, 0.1% SDS, 1% Triton X-100, 1 mM EDTA, 150 mM NaCl, 10 mM Tris HCl, pH 7.5), TX-100 (0.75% Triton X-100, 0.5 M NaCl, 1 mM EDTA, 10% glycerol, 20 mM Tris HCl, pH 8) and NP-40 (0.5% NP-40, 5 mM MgCl2, 120 mM NaCl, 5 mM Tris HCl, pH 7.5). All lysis buffers contained 2 mM PMSF and 10 µl/ml protease inhibitor cocktail (Sigma). Extracts were stored at −20°C.
Protein content was measured by protein precipitation with UPPA-1 and UPPA-2 (Geno Tech, St. Louis, MO) according to the manufacturer's directions; re-solubilization (0.1% SDS, 1% deoxycholate, 0.5M NaOH), followed by the Lowry method 
. BSA was used for the standard curve.
Quantitative Western Blotting
Western blotting was done as described previously 
. The primary antibody was 1.5 µg α-cyclin B1 clone GNS1 (PharMingen) and the secondary was 5 µl alkaline phosphatase conjugated goat-α-mouse IgG (Promega, Madison, WI) in 15 ml blocking buffer. The substrate was CDP-star (Tropix, Bedford, MA). Blots were imaged with a Flour-S MultiImager (BioRad) and images analyzed with Quantity One software (BioRad).
Cell Fixation and Lyophilization
Single cell suspensions were fixed in 0.5% formaldehyde for 10 min at 37°C followed by 90% methanol at −20°C. Fixed cells were lyophilized as individual samples in 1.5 ml centrifuge tubes on a Labconco Freezone 12 Liter Freeze Dry System, model 77540 (Kansas City, MO) using histidine lyophilization buffer (5 mM histidine, 0.1% Tween 20, 2 mM sucrose). Lyophilized samples were stored in desiccators at −80°C. Samples were rehydrated with distilled water equal to the amount of lyophilization buffer used.
Samples were stained for cyclin B1 and DNA as previously described 
. Briefly, 5×105
cells were incubated with 0.25 µg GNS1, followed by washing, then incubated with 1.25 ug FITC-conjugated goat anti-mouse IgG (1.25 µg). RNA was digested with RNase and then propidium di-iodide was added at a final concentration of 50 µg/ml. Cells were analyzed on a Coulter EPICS XL- MCL Cytometer (Coulter Electronics, Miami, FL). In some experiments, cells were stained with GNS1 conjugated to Alexa Fluor 647 and phycoerythrin conjugated cyclin A2 antibody (gift from Vince Shankey, Beckman Coulter, Miami, FL); DNA was stained with DAPI, and cells were analyzed on a BD LSR I Cytometer (BD Biosciences, San Jose, CA).
All data primary analysis was performed offline with WinList 6.0 (Verity Software House, Topsham, ME), and non-linear regression was performed with GraphPad 5.0, GraphPad Software, San Diego California USA).
Background Subtraction: To reduce background to near an average of zero, we subtracted median fluorescence as a function of cyclin B1 expression for G1 cells. Since cyclin B1 is not expressed for much of G1, this provided a near perfect control for background binding of the GNS1 antibody. This was accomplished in WinList (Verity Software House, Topsham, ME) using compensation algorithms.
Calculation of molecules per cell
Optical densities (OD) from CCD exposure to Western blot chemiluminescence from known numbers of cyclin B1-expressing cells and from a serial dilution of purified recombinant cyclin B1 were used to determine the average molecules of cyclin B1 per cell. Cytometry was used to obtain the average cyclin B1-related fluorescence per cell for the same cell lines (duplicate cultures) used in the Western blots. Linear regression was used to relate OD to molecules and fluorescence to molecules for the standard and test cell lines 
Calculation of cell cycle expression of cyclin B1
After background subtraction, gates on cyclin A2 versus light scatter, DNA content versus light scatter, and cyclin B1 versus DNA content were used to isolate only the 2C cycling component from G1 through metaphase. Contiguous arbitrary regions were set on cyclin B1 versus DNA content bivariate histograms from early G1 through G2+M. Mean cyclin B1 levels per region were plotted versus a cumulative function (1) of the cell frequency per region.
the frequency of cells within a region; i
the region index beginning at 0 and progressively moving through the cell cycle.