Dear Hani Alrefai!

Here is the MACs separation of permeabilized fixed cells. Please chek it and came back to me

in case of some conflicts.

Sincerely yours!

Dr. Bratko Filipič

Email: [email protected]

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ABSTRACT

We have developed a two-layer density gradient in which the epithelial cells form a single density band. This was demonstrated by recovery experiments using [3H]thymidine-labelled epithelial cells which showed epithelial cells were enriched within this first step by a factor of 20. In a second step the h4ACS system was applied. Cells were stained with a preformed FITC-conjugated mouse anti-human cytokeratin antibody bound to a rat anti-mouse antibody coupled to superparamagnetic particles (immune paramagnetic separation complex; IPSC) and subjected to high gradient magnetic fields. The two-step procedure was confirmed by dispersing 50 epithelial cells in 5 x lo’, 5 x 106, 5 X 107, 5 X 108, 5 X lo9 peripheral blood leucocytes. Specific binding of the preformed IPSC was demonstrated by flow cytometry, confocal laser, fluorescent and electron microscopy. The specificity of the method was further proved by dual staining with IPSC and anti-human PSA antibody of epithelial prostatic cells separated from peripheral blood in vitro. By means of this double-step separation method it was possible to isolate up to 15-20 cells out of 50 epithelial cells originally suspended into 5 X 10’ to 5 X 10” human peripheral blood leucocytes. This represented an enrichment factor between 20 000 and 200 000, depending on the initial cell number. The immunologically captured epithelial cells can be used for further cytogenetic investigations such as in situ hybridization (ISH)

2. Materials and methods

2.1. Cell culture

The MCF-7 (ATCC, HTB 22) and SK-BR-3 (ATCC, HTB 30) cell lines were grown in DMEM and McCoy’s medium respectively with 10% fetal calf serum (FCS), 5 mM r_-glutamine, l/100 dilution of Sigma A 9909 (10000 U penicillin/lo mg streptomycin/25 pg amphotericin B in 0.9% sodium chloride) (Sigma-Aldrich, 82039-Deisenhofen, Germany). Freshly resected prostatic cancer tissue was minced and subsequently filtered through a cell strainer (Falcon, Becton Dickinson, 6900 Heidelberg, Germany).

2.2. Preparation of the density gradient and centrifugation medium

12 ml polystyrol tubes, saturated with 1% FCS in PBS for at least 2 h, were filled with 3 ml PolymorphPrep (Immune, Heidelberg, Germany, d = 1.113 g/ml, osmolality = 460 mosM) and carefully overlaid with 3 ml NycoPrep 1.068 (Immuno, Heidelberg, Germany, d = 1.068 g/ml, osmolality = 335 mosM). At least 5 ml EDTA treated blood was laid on top of the gradient. The tube was centrifuged at 450 x g in a bench centrifuge (Sigma 3E-1, Sigma, Germany) for 20 min at room temperature. The single cell band between the interface of the platelet enriched plasma and the density of 1.068 g/ml was removed cautiously by a small syringe (canule: 1.2 mm diameter, 18 gauge). Removed cells were pelleted by centrifugation at 200 x g.

2.3. [3H]thymidine-label assay for the determination of the buoyant density of epithelial / tumour cells

Prior to the incubation of 5 x lo7 MCF-7 cells with [ 3H]thymidine ([6- 3H]thymidine, specific activity 0.74 Tbq/mmol (Amersham LifeScience, England) for 12 h under sterile conditions with gentle agitation, the MCF-7 cells were filtered through a cell strainer (Falcon, Becton Dickinson, 6900 Heidelberg, Germany) to remove doublets or higher cell aggregates. The cells were washed three times with 40 ml phosphate buffered saline (PBS) and centrifuged at 200 X g. The pellet was suspended in 1 ml PBS. The total activity of the cell suspension was 2.9 kBq. Finally the cells were mixed with 40 ml EDTA treated peripheral blood. A volume of 5 ml of the blood/[ “Hlthymidine incubated MCF-7 cells suspension was carefully transferred to the top of a 6 ml density gradient medium. The gradient tubes (polystyrol, diameter 16.8 mm, height 100 mm, volume 12 ml; Greiner, Solingen, Germany) were centrifuged for 20 min (450 X g> at room temperature in a bench centrifuge (Sigma 3E-1, Sigma, Germany). For the measurement of the radioactivity the gradient was transferred in 500 ~1 portions into scintillation vials containig 5 ml lysis buffer (75 mM NaCl, 25 mM Na,EDTA, pH 8.0). The scintillation cocktail (15 ml) was added (QuickSzint 212, Zinsser, Frankfurt, Germany) and the [ 3H]thymidine p-activity was measured in a beta counter (Rachbeta 1274, LKB Wallac, Finland).

2.4. Fixation procedure

The cells obtained from the single band at the interface of the platelet enriched plasma and the 1.068 NycoPrep density medium were resuspended in 1 ml PBS/l% FCS and fixed in 1 ml of an 8% paraformaldehyde/PBS fixative (pH 7.4) reaching a final concentration of 4% paraformaldehyde/PBS for at least 2 h at room temperature. The fixed ceil suspension could be stored at 4” C. 2.5. Immunocytochemical staining Preformed complexes and incubation Prior to the cell staining a complex was preformed between the FITC-conjugated mouse anti-human cytokeratin 8/18 antibody (50 ~1) (final antibody concentration: 5 pgg/ml; clone CAM5.2; Ig chain composition: IgG2a heavy chain and K light chains; Becton Dickinson, Heidelberg, Germany) and the rat anti-mouse IgG2a + b antibody coupled to microbeads (25 ~1) (final antibody concentration: 2.5 pg/ml; Miltenyi, 51429 Bergisch Gladbach, Germany) by preincubation (immune paramagnetic separation complex, IPSC) at 4°C for 15 min. The preformed IPSC was purified from non-bound mouse antihuman cytokeratin antibody using a MiniMACS column (Miltenyi, 51429 Bergisch Gladbach, Germany). A volume of enriched 175 ~1 cell suspension, obtained by the density gradient separation, was incubated with the preformed IPSC (75 ~1). The incubation medium contained PBS/0.3%: (v/v> saponin (Sigma-Aldrich, 82039 Deisenhofen, Germany; saponin from Saponaria species) and 0.5% fetal calf serum (Boehringer, Mannheim, Germany). After an incubation time of 15 min at room temperature and 30 min at 4” C the cell suspension was washed twice in 1 ml PBS/0.3% (v/v) saponin.

Detection of preformed IPSC

For the detection of intracytoplasmic staining of the IPSC we used a biotinylated polyclonal rat anti-mouse IgG F(ab’>, antibody (final concentration: 5 pg/ml; Dianova, 20011 Hamburg, Germany) bound to fluorescent streptavidinphycoerythrin (SA-PE) (final concentration: 5 pg/ml) (Dianova , 20011 Hamburg, Germany).

2.6. Controls for nonspecific binding

Anti-isotype antibody The nonspecific binding of the IPSC was determined by an anti-isotype control antibody. The antibody (clone X39; final concentration 5 kg/ml; Ig chain composition: IgG2a, FITC conjugated; Becton Dickinson, Heidelberg, Germany) is specific for keyhole limpet hemocyanin (KLH), an antigen not expressed on human cells or human cell lines. The rat anti-mouse IgG2a + b coupled to microbeads was bound to the isotype control antibody using the same procedure as described for the IPSC. 50 MCF-7 cells diluted in 5 X 10’ leucocytes (final staining volume: 250 ~1) were used as a nonspecific binding control. Rat anti-mouse antibody coupled to microbeads The nonspecific binding of the applied rat anti-mouse IgG2a + b antibody coupled to microbeads as a constituent of the IPSC was investigated by incubating 50 cancer cells (MCF-7) in 5 X 10’ leucocytes (225 ~1) with 25 ~1 of only the rat anti-mouse IgG2a + b antibody coupled to microbeads (final dilution: l/10) reaching a final volume of 250 ~1.

2.7. Titration of the IPSC

The titration of the IPSC was accomplished as follows: the complex was preformed and subsequently applied to the epithelial cells (MCF-7). The IPSC was applied in ten concentration steps ranging from 0.005 pg/ml to 10 pg/ml. Titration was determined by flow cytometry using the differences of the mean channel of cytokeratin-FITC signals of positive cells. The evaluation of the IPSC titration-signals were accomplished in histogram acquisition mode comparing the mean channel intensities.

2.8. IPSC used for cell separation

The magnetic cell sorter (MACS) consists of a permanent magnet into which different sizes of separation columns can be fitted. The separation columns are made of polystyrene, differing in size, and are packed with steelwool fibres working primarily as an efficient cell binding surface. The steelwool fibres also function as a dielectric producing high magnetic gradients in the permanent magnet system. The magnetic field strength has been calculated to be 0.6 Tesla by the manufacturer (Miltenyi et al., 1990). The column was prepared for the separation run as described in the literature. In brief, the column was rinsed from bottom to top once with -20” C cold 70% methanol, then from top to bottom with 30 ml PBS/l% FCS. The column was saturated with 1% FCS for 2 h. The separation experiments were carried out with 50 epithelial cells, dispersed in 1 ml of 5 X 105, 2 ml 5 X 106, 5 ml 5 X 107, 10 ml 5 X lo’, 20 ml 5 x lo9 leucocytes. The dilutions were tested for separation efficiency using three flow rates on the column, as determined by the size of the canulae. We used 24 gauge (0.55 mm diameter, flow rate: 0.6 ml/min), 20 gauge (0.8 mm diameter, flow rate: 3.5 ml/min) and 18 gauge (1.2 mm diameter, flow rate: 12 ml/min) canulae. The cells were pipetted in aliquots of 500 ~1 onto the top of the column and followed through by a 4 fold excess of PBS/FCS relative to the original cell suspension (i.e. 2 ml). The IPSC targeted ‘magnetic MCF-7 cells’ were bound within the magnetic field to the steelwool fibres while negative, ‘non-magnetically stained’ cells passed the column and were collected in a tube as the ‘non-magnetic’ fraction. This negative eluate was subsequently reapplied to the column four times. The column was then removed from the MACS stand and the cells flushed sidewards from the bottom to the top with a volume of 2 ml PBS/FCS using a syringe connected to a threeway stopcock. The separation column was replaced again in the MACS stand. Nonspecifically attached cells passed through the column by gravity until the outflow from the column was terminated by the surface tension of the elution buffer. This procedure was repeated twice. Finally the column was rinsed with 2 ml PBS/l% FCS to remove the last nonspecifically attached cells.

2.9. Removal of the magnetically bound fraction from the column

The three-way stopcock was closed and the canula removed. A 10 ml syringe filled with PBS/l%FCS buffer was connected to the top of the column and then removed from the MACS stand. The ‘positive magnetic fraction’ was flushed from the top to bottom into a 12 ml tube (polypropylene; size: 17 X 20 mm; Becton Dickinson, Heidelberg, Germany).

2.10. Fluorescence microscopy

Cell staining of the IPSC was monitored using a fluorescence microscope (Leitz Laborlux 12 microscope; Leitz excitation filter BP 515-560; objectives: Plan Neofluor 63 and 40; Leitz Wetzlar, Germany).

2.11. Confocal laser analysis of the IPSC stained cells

After rinsing with PBS, specimens of interest were mounted with 90% glycerol/lO% PBS supplemented with 0.2% phenylenediamine and viewed at a Leitz epifluorescence microscope equipped with a confocal imaging system MRC- 500 (Bio-Rad Microscience, Munich, Germany; excitation filter 488 DFlO; dichroic reflector 510 LP; emission filter OG 515 LPI. Photographs were taken with a Kodak TMY 400 film with a NIKON reflex camera from a black/white monitor.

2.12. Flow cytometry

The IPSC bound to the cells were counter stained with propidium iodide (PI) and were subjected to flow cytometry analysis. For cell analysis a Becton-Dickinson FACScan Analyser, equipped with an argon laser arrangement (excitation wavelength A = 488 nm), was used. The data acquisition mode was forward- and side-scatter (linear amplification), fluorescence of PI and fluorescence of FITC (fluorescein isothiocyanate) (logarithmic amplification). The IPSC-separated prostatic epithelial cells were counter-stained using the mouse anti-human PSA antibody (monoclonal mouse anti-human prostate specific antigen, clone ER-PR8; Ig chain composition: IgGl, DAK0 Diagnostika GmbH, Hamburg, Germany) detected by a phycoerythrin (PE)-conjugated goat anti-mouse antibody (polyclonal goat anti-mouse, Ig chain composition: IgGl, R-phycoerythrin conjugate; Medac, Hamburg, Germany).

2.13. Transmission electron microscopy (TEM)

For transmission electron microscopy, samples were fixed with Karnowsky’s reagent at 4°C for 24 h and then washed with PBS. The samples were post-fixed with 1.3% 0~0, in 0.1 M collidine buffer, pH 7.3, and washed with PBS before dehydration with ethanol and embedding in Epon 812. Ultra-thin sections were mounted on 200- mesh copper grids. The specimens were examined with a Philips EM 410 at 60 kV.

Thanks Filipic

Thanks Christopher