3 Material
3.4 Cell lines
Cell line Desription
Hek 293T human epithelial kidney cells, transformed
by SV 40 T antigen, semi-adherent
N2a mouse neuroblastoma cells, adherent
H6 clone H6 of mouse neuroblastoma cells,
adherent, highly susceptible for scrapie infection [179]
BCA Reagent B Pierce, Bonn, Germany
Ethylene glycol tetraacetic acid (EGTA) Sigma-Aldrich, Deisenhofen, Germany Ethylenediamine-tetraacetic acid disodium
Material 28
Poly-L-lysine hydrobromide (cat. no. P1399) Sigma-Aldrich, Deisenhofen, Germany
Ponceau S Roth, Karlsruhe, Germany
KpnI, 10 U/µl NEB, Frankfurt am Main, Germany
MluI, 10 U/µl MBI Fermentas, St. Leon-Rot, Germany
MspI, 10 U/µl MBI Fermentas, St. Leon-Rot, Germany
SacI, 10 U/µl MBI Fermentas, St. Leon-Rot, Germany
SalI, 10 U/µl MBI Fermentas, St. Leon-Rot, Germany
Calf Intestine Alkaline Phosphatase (CIP), 10 U/µl
NEB, Frankfurt am Main, Germany Phusion Polymerase, 2 U/µl Finnzymes, Espo, Finland
PNGaseF (Peptide: N-Glycosidase F), 500 U/µl
NEB, Frankfurt am Main, Germany T4-Ligase, 5 U/µl MBI Fermentas, St. Leon-Rot, Germany T4-DNA-Polymerase, 5 U/µl MBI Fermentas, St. Leon-Rot, Germany Proteinase K, 30 U/mg, cat. no. P2308 Sigma-Aldrich, Deisenhofen, Germany
Proteinase K, 5-15 U/mg, cat. no. P8044 Sigma-Aldrich, Deisenhofen, Germany Qiaquick Gel Extraction Kit, cat. no. 28706 Qiagen, Hilden, Germany MinElute Reaction Cleanup Kit, cat. no.
28204
Qiagen, Hilden, Germany
3.9 Loading buffers for proteins:
5 x Laemmli loading buffer 100 mM Tris-HCl, pH 8.0 12.5 % SDS
25 % β-Mercaptoethanol 5 % Glycerol
bromphenol blue for DNA:
10 x DNA loading dye 1 mg/ml Bromphenol blue
50 % Sucrose 1 mM EDTA 3.10 Marker
Description Source
MassRuler DNA Ladder Mix MBI Fermentas, St. Leon-Rot, Germany GeneRuler 1kb DNA Ladder MBI Fermentas, St. Leon-Rot, Germany Prestained Protein Molecular Weight Marker MBI Fermentas, St. Leon-Rot, Germany Biotinylated SDS-PAGE Standard Broad
Material 30 OptiMEM, cat. no. VX31985054 Invitrogen (Gibco), Karlsruhe, Germany for prokaryotic cells:
pWPT2264f, forward primer for sequencing AAC CGG TGC CTA GAG AAG GT pWPT3415r, reverse primer for sequencing GAA AAT GAA AGC CAT ACG GG 3.13 Plasmids
B. Thaa, Molecular Toxicology, Konstanz, Germany
pWPT-GFP, transfer plasmid for lentiviral gene transfer
psPAX2, packaging plasmid for lentiviral gene transfer
D. Trono, School of Life Science, Lausanne, Switzerland
pMD2.G, envelope plasmid for lentiviral gene transfer
D. Trono, School of Life Science, Lausanne, Switzerland
pEGFP-N1 BD Bioscience, Heidelberg, Germany
pCMS-PrP-EGFP S. Ramljak, University Hospital Göttingen, Germany
3.14 Software
Softare Supplier
AIDA Image Analysis Software 3.10 Raytest, Straubenhardt, Germany AxioVision AxioVs40 V4.3.0.101 Zeiss, Oberkochen, Germany
CarelDraw 12 Corel Corporation
Clone Manager 7 Scientific and educational software Cary, NC, USA
EMBOSS http://www.ebi.ac.uk/emboss
EndNoteX Thomson ISI Research Soft
Graph Prism 5.0 GraphPad Software Inc
ImageJ 1.39 Wayne Rasband, National Institute of
Health, USA
EDTA solution for trypsin-free detachment of eukaryotic cells
Freezing medium for eukaryotic cells 70 % growth medium with supplements 20 % FCS
10 % DMSO
Freezing medium for prokaryotic cells 25 % Glycerin (100 %)
Material 32 10 x Laemmli running buffer for SDS-PAGE 250 mM Tris
1.92 M Glycine
Sucrose-Relaxation buffer 270 mM Sucrose
Substrate solution for Cell ELISA 50 mM Glycin-NaOH pH 9.7 4 mM MgCl2
Centrifuges Centrifuge 5810R (Eppendorf, Hamburg,
Germany)
Cell counter Casy TT Schärfe System, Reutlingen, Germany Cell culture plastic ware Corning, Schiphol-Rijk, Netherlands Centrifuge tubes (15, 50 ml) Corning, Schiphol-Rijk, Netherlands Centrifuge tubes, thick wall for
ultracentrifugation, cat. no. 355631
Beckman, Krefeld, Germany
Material 34
Coverslips Menzel, Braunschweig, Germany
Documentation of agarose gels Gel Jet Imager (Intas, Göttingen, Germany)
Electrophorese chambers for DNA:
Agagel Mini Biometra (Biomed, Göttingen, Germany) Luminescence Image station LAS 1000 Pro (Fujifilm, Düsseldorf, Germany)
Lumox dishes Greiner-bio-one, Frickenhausen, Germany
Microscopes Confocal microscope: LSM 510 Meta (Zeiss,
Oberkochen, Germany)
Fluorescent microscopes: Axiovert S100 TV and 200M (Zeiss, Oberkochen, Germany)
Object slides Menzel, Braunschweig, Germany
Photometer Photometer 2100 pro (Amersham
Biosciences, Freiburg, Germany)
Pipettes Abimed, Langenfeld, Germany
Pipette tips Sarstedt, Nümbrecht, Germany
Power supplies Model 200 (BioRad, München, Germany)
Power Pac 300 (Amersham Biosciences, Freiburg, Germany)
Reaction tubes (0.5-1.5 ml) Sarstedt, Nümbrecht, Germany
Thermomixer Thermomixer comfort (Eppendorf, Hamburg,
Germany
Transfer membrane PVDF membrane Hybond P (Amersham
Biosciences, Freiburg, Germany)
Vortex Genie 2 VWR, Darmstadt, Germany
4 Methods
4.1 Cell culture
4.1.1 Culture of eukaryotic cells
N2a cells and the N2a cell clone H6 were cultured in DMEM w/o pyruvate, supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine and 10 % heat-inactivated FCS. H6-22L cells, scrapie-infected cells, were cultured in OptiMEM with the above mentioned supplements. HEK293T cells were maintained in DMEM with pyruvate with the above described supplements. All cell lines were kept at 37 °C and 5 % CO2. They were passaged regularly. In case of H6-22L the passages had to be performed every 2nd o 3rd day in a ratio of 1:2 or 1:3, so that the scrapie infection would not be lost rapidly.
4.1.2 Cryopreservation of eukaryotic cells
The cells were harvested, counted, centrifuged (1,000 rpm (Labofuge 400, Heraeus, Hanau, Germany), 5 min) and resuspended in ice-cold freezing medium at a cell density of 106-107 cells/ml. The cells were transferred into cryo vials and stored overnight in a cryo contrainer containing isopropanol at -80 °C for gentle freezing of the cells The next day, vials were transferred and stored in liquid nitrogen until use. Thawing of the cells was performed as follows: The cells were thawed at 37 °C in a water bath and directly transferred into 20 ml growth medium to dilute the DMSO. The cells were spun down (1,000 rpm (Labofuge 400, Heraeus, Hanau, Germany), 5 min) and resuspended in the appropriate growth medium.
4.1.3 Transient transfection of N2a, H6 or H6-22L with Lipofectamine2000
For immunofluorescence (IF) 2 x 105 cells were seeded on poly-L-lysine coated coverslips.
The next day the cells were transfected with 0.5 µg DNA and 2 µl Lipofectamine. For protein analysis 15 cm dishes were transfected in a ratio of 20 µg DNA and 40 µl Lipofectamine. The protocol was performed according to the manufacturer´s instruction. A change of the medium after 6 h was not necessary. A transfection control with pEGFP-N1 was always included. The transfection efficiency was between 30-60 %. After 24 to 48 h the cells were fixed and stained for IF or in case of protein analysis the cells were lysed after 72 h.
Methods 36
4.1.4 Infection of H6 cells with scrapie strain 22L
The highly susceptible clone H6 of N2a cells was infected with the 22L strain (kind gift of Prof. S. Lehmann, Montpellier, France) as described previously [179], leading to a persistent infection. Such cultures were termed H6-22L.
As freezing of H6-22L and culturing over 3 months led to a loss of infection, it was necessary to infect regularly a new passage of H6 cells with the scrapie strain 22L. Because cell culture medium of H6-22L cells also contains PrPSc, it is possible to infect H6 cells with medium. For this purpose 103 and 104 H6 cells were seeded on 48 well dishes. Meanwhile the cell culture medium from already scrapie infected H6-22L cells was harvested, centrifuged at 1,000 rpm (Labofuge 400, Heraeus, Hanau, Germany) for 5 min and the supernatant was diluted 1:2 with fresh growth medium. The next day the medium from H6 cells grown in 48 well dish was replaced by the 1:2 diluted growth medium from H6-22L cells. The H6 cells were grown to confluency and transferred to a 12 well dish. After growing to confluency the cells were transferred to 25 cm2 flask. Here, the cells were passaged every 2nd or 3rd day 1:2 or 1:3.
Already after 3-4 weeks post infection the scrapie infection could be monitored by IF or cell ELISA.
4.2 Immunocytochemistry
4.2.1 Immunofluorescence staining
4.2.1.1 Coating of coverslips or lumox dishes with poly-L-lysine (PLL)
Sterile PLL (4 mg/ml PBS) was used in an end concentration of 0.2 mg/ml PBS. The lumox dishes or coverslips were incubated with PLL for 1 h at RT or 30 min at 37 °C. After that the dishes or coverslips were washed 3 x with PBS for 5 min and could be used immediately for seeding of cells.
4.2.1.2 Staining of PrPSc
To monitor the status of the scrapie infection of the newly infected and in already infected H6-22L cells, PrPSc was stained in these cells and the PrPSc positive cells were counted afterwards.
H6-22L cells were seeded on glass coverslips to semiconfluency in a 12 well dish. They were fixed with 4 % formaldehyde solution (FA), freshly prepared from paraformaldehyde (PFA), for 20 min at RT. After pemeabilisation of the cells with 0.4 % Triton X-100 in PBS for 3 min, the cells were denatured with 6 M GdnHCl in 50 mM Tris-HCl pH 7.4 for 10 min at RT to allow the antibody (Ab) to bind PrPSc The unspecific binding sites for the Ab were blocked with 1 % BSA/PBS for 30 min at RT. The incubation with the 1st Ab 6H4 1:5,000 or Kan72 1:500 diluted in 1 % BSA took place in a humid chamber for 30 min at 37 °C. Alternatively, the incubation with the 1st Ab took place o/n at 4 °C. The coverslips were washed 3 x for 5 min with PBS before incubation with the fluorochrome labelled 2nd Ab gαm A488 or gαr A488 diluted 1:1,000 for 30 min at 37 °C in a humid chamber. The cells were washed 1 x with PBS and then stained with 200 ng/ml Hoechst 33342 for 5 min at RT. After washing 3 x for 5 min with PBS the cells were embedded with Aqua Poly Mount.
4.2.1.3 Induction and staining of lipid bodies (LBs)
N2a or H6-22L cells were seeded on coverslips. After attachment of the cells to the coverslips (approx. 6 h later) the production of LBs was induced by addition of 200 µM oleic acid directly to the medium. After 16 h of incubation with oleic acid LBs were visible in light microscopy as big, round vesicles in the periphery of the cells. The cells were fixed with 4 % FA for 20 min at RT, permeabilised with 0.4 % Triton X-100 for 3 min at RT. Then the cells were blocked with 1 % BSA for 30 min at RT, incubated with the 1st and 2nd Abs and then stained for LBs with 1 µg/ml Bodipy 499/508 in PBS for 15 min at RT. The cells were washed 1 x with PBS and then stained with 200 ng/ml Hoechst 33342 for 5 min at RT. After washing 3 x for 5 min with PBS the cells were embedded with Aqua Poly Mount.
4.2.1.4 Concanavalin A (ConA) stimulation
To observe a possible endocytosis of plasma membrane bound proteins, the internalisation process was driven by ConA. ConA-A594 was diluted to 1 µg/ml in DMEM and cells were incubated for 10, 30 or 100 min at 37 °C. Unlabelled ConA was dissolved in PBS supplemented with 1 mM MgCl2 and 1 mM MnCl2 to a stock solution of 50 mg/ml. Cells were incubated for 10, 30 or 100 min at 37 °C with 100 µg/ml unlabelled ConA diluted in DMEM prior to fixation.
Methods 38
4.2.1.5 Staining of PrPSc in combination with trafficking organelles
N2a or H6-22L cells were seeded to semiconfluency on glass coverslips. In some cases the cells were treated with unlabelled ConA prior to fixation. The cells were fixed with 4 % FA and simultaneously permeabilised with 0.1 % digitonin for 1 h at RT. In case of H6-22L, a denaturation step with 6 M GdnHCl for 10 min followed. After blocking with 1 % BSA for 30 min at RT, the cells were incubated with the primary Ab 6H4 1:1,000 or Kan72 1:500 diluted.
Subsequently, the cells were incubated with the secondary Ab gαm-A488 or gαr-A488 each diluted 1:1,000. Afterwards the incubation with the Abs against the trafficking organelles followed: 1st Ab gαEEA1 1:200, gαLimp2 1:50, rαERp29 1:500 or mαGM130 1:1000. The 2nd Abs were dαg-Cy3, gαr-A546, gαm-A546, each 1:1,000 diluted. All Abs were diluted in 1 % BSA and incubated for 30 min at 37 °C in a humid chamber. After each Ab incubation the cells were washed 3 x for 5 min with PBS. The nuclei were stained with 200 ng/ml Hoechst 33342 for 5 min at RT. After washing 3 x for 5 min with PBS the cells were embedded with Aqua Poly Mount.
4.2.1.6 Assay for the detection of PrPSc at the plasma membrane
2 x 105 N2a or H6-22L cells were grown on lumox dishes in a 24 well format, pretreated with PLL for 1 h at RT. 24 h later the cells were fixed and permeabilised with 8 % FA/0.1 % GA and 0.1 % digitonin for 1 h at RT. Cells were incubated with proteinase K (PK) in an end concentration of 20 µg/ml, diluted in PBS, for 15 min at 37 °C. The digestion was stopped with 2 mM PMSF, diluted in PBS, for 15 min at RT. In case of H6-22L, the cells were denatured with 6 M GdnHCl for 10 min. After blocking with 1 % BSA for 30 min at RT, the cells were incubated with primary and secondary Abs (6H4 1:1,000; gαm-A488 1:1,000) diluted in 1 % BSA for 30 min at 37 °C. In some cases the cells were also stained for the plasma membrane marker cholera toxin subunit B-A555 (CTB) in a dilution of 0.1 µg/ml for 10 min at RT. The nuclei were stained with 200 ng/ml Hoechst 33342 for 5 min at RT. The lumox membrane with the cells was cut out and embedded with Aqua Poly Mount on glass object slides.
4.2.1.7 Cell ELISA
N2a and H6-22L cells were seeded to confluency on a 96 well plate. The next day the cells were fixed with 4 % FA for 1 h at RT. After washing with PBS the cells were permeabilised with 0.4 % Triton X-100 for 3 min at RT. Then the cells were digested with 20 µg/ml PK (P8044) for 15 min at 37 °C. The reaction was stopped with 2 mM PMSF for 15 min at RT. The denaturation was performed with 6 M GdnHCl for 10 min at RT. After washing 3 x with TNT buffer the cells were blocked with 5 % milk powder/TNT buffer (blocking solution) for 1 h at RT. The 1st Ab Kan72 1:1,000, 6H4 1:5,000 or 15B3 1:100 were diluted in blocking solution and incubated for 1 h at RT or o/n at 4 °C. The cells were washed 3 x in TNT buffer and then the incubation with the 2nd Ab gαr-AP 1:2,000, gαm-AP 1:2,000 or gαm IgM-AP 1:10,000 diluted in blocking solution followed for 1h at RT. The cells were washed 3 x with TNT, followed by a washing step with 50 mM glycine-NaOH pH 9.7. Then the cells were incubated with the substrate solution until a staining in H6-22L or an unspecific staining of N2a cells appeared. The substrate solution contained BCIP and NBT. BCIP is the alkaline phosphatase (AP) substrate. After its dephosphorylation with AP, BCIP is oxidised to a dark-blue indigo-dye. NBT serves herein as the oxidant and is reduced itseld to a dark-blue indigo-dye.
4.2.1.8 Modification of the cell ELISA
To test other Abs (15B3, V5B2) for specific staining of PrPSc, the cell ELISA protocol was modified. The basic changes were the permeabilisation with 40 µg/ml digitonin for 3 min at RT, 0.5 % saponin, 12 min at RT or 0.2 % sarcosyl, 10 min, RT and staining with the 1st Ab 15B3 1:100 – 1:500 diluted in growth medium without supplements or 1 %BSA prior to fixation. Also the blocking solutions were modified from 5 % milk powder/TNT, 1 % BSA, 10
% mouse serum or 10 % normal goat serum (NGS). Another variance lay in the change of washing buffers from TNT, PBS to high salt washing solution.
Methods 40
4.2.2 Immunostaining for electron microscopy
4.2.2.1 Staining of PrPC and trafficking organelles in H6-22L cells upon crosslinking with antibodies
5 x 106 H6-22L cells were seeded on 6 cm lumox dishes, pretreated with PLL. The next day the medium was discarded and the cells were washed 2 x with PIPES (0.1 M PIPES pH 7.2).
The mAb 6H4 was diluted 1:500 in PIPES and the cells were incubated in vivo with the Ab for 120 min at 4 °C. The cells were washed 3 x with PIPES for 1 min. The 2nd Ab gαm F(ab)2 Au10 nm was diluted 1:70 in PIPES and incubated for 0, 15, 60 or 180 min at 37 °C. After washing the dishes for 5min with PIPES, the cells were fixed and simultaneously permeabilised with 8
% FA/0.1 %GA first for 5 min, then in the same fixative supplemented with 0.1 % digitonin in PIPES for 55 min at RT. Cells were washed 3 x for 5 min with PIPES. PK digestion (20 µg/ml in PIPES) was performed for 15 min at 37 °C. The digestion was stopped with 2 mM PMSF in PIPES for 15 min at RT. The denaturation step was performed in an ascending and descending sequence in 1-molar steps from 1 M to 6 M GdnSCN, each step with 5 min duration. Samples were dehydrated with increasing ethanol series (30, 50, 70, 90, 95, 100
%), each for 30 min, before embedding in the methacrylate resin LR-Gold. The embedding was as follows: LR-Gold:Ethanol (1+1) o/n at 4 °C, LR-Gold for 3 h on ice, LR-Gold with 0.1 % benzil 2 x for 2h on ice. Afterwards the lumox membrane was cut into small pieces. For polymerisation the lumox pieces were put into 1.5 ml reaction tubes. The polymerisation took place by UV light first for 72 h at -30 °C (the temperature in the samples was risen to -4
°C due to exothermic reaction), then for 24 h at RT. Ultrathin sections were cut at the microtome and collected on Formvar-coated Nigrids. Some sections were stained with Limp2 and pA-Au5nm (post-embedding labelling).
4.2.2.2 Staining of PrPSc and trafficking organelles in H6-22L
5 x 106 H6-22L cells were seeded on 6 cm lumox dishes, pretreated with PLL. The next day the medium was discarded and the cells were washed 2 x with PIPES (0.1 M PIPES pH 7.2).
The cells were fixed and simultaneously permeabilised with 8 % FA/0.1 %GA first for 5 min, then in the same fixative supplemented with 0.1 % digitonin in PIPES for 55 min at RT. Cells were washed 3 x for 5 min with PIPES. PK digestion (20 µg/ml in PIPES) was performed for 15 min at37 °C. The digestion was stopped with 2 mM PMSF in PIPES for 15 min at RT. The
denaturation step was performed in an ascending and descending sequence in 1-molar steps from 1 M to 6 M GdnSCN, each step with 5 min duration. Afterwards the cells were incubated with the mAb 6H4 1:1,000 in 1 % BSA-c/PIPES o/n at 4 °C. Then the cells were washed 3 x for 5 min with PIPES and stained with the 2nd Ab gαm Au5nm 1:50 for 1h at 37 °C.
The cells were washed again 3 x for 5 min with PIPES, before they were dehydrated and embedded as described above. Section labelling was done with EEA1 and pA-Au10nm or Limp2 and pA-Au10nm. Then the cells were stained again with Ab 6H4 and 2nd Ab gαm Au5nm.
4.2.2.3 Staining of PrPSc and trafficking organelles in H6-22L upon treatment with ConA 5 x 106 H6-22L cells were seeded on 6 cm lumox dishes, pretreated with PLL. The next day the cells were pretreated with 100 µg/ml unlabelled ConA for 10, 30 or 100 min at 37 °C.
Afterwards, the cells were washed 2 x with PIPES (0.1 M PIPES pH 7.2), fixed and simultaneously permeabilised with 8 % FA/0.1 %GA first for 5 min, then in the same fixative supplemented with 0.1 % digitonin in PIPES for 55 min at RT. Cells were washed 3 x for 5 min with PIPES. PK digestion (20 µg/ml in PIPES) was performed for 15 min at37 °C. The digestion was stopped with 2 mM PMSF in PIPES for 15 min at RT. The denaturation step was performed in an ascending and descending sequence in 1-molar steps from 1 M to 6 M GdnSCN, each step with 5 min duration. After embedding, as described above, post embedding labelling was performed with Ab 6H4 and gαm-Au10 nm, EEA1 and pA-Au5nm or Limp2 and pA-Au5nm.
4.3 Working with lentiviruses
4.3.1 Transfection of HEK293T for production of recombinant lentiviruses
In a 10 cm dish 4 x 106 HEK293T were seeded. After 24 h the cells were transfected with CaPO4. For the production of lentiviruses 15 µg pWPT with the appropriate insert, 10 µg psPAX2 and 5 µg pMD2.G were resuspended in 400 µl sterile H2O. After addition of 100 µl 2.5 M CaCl2 the solution was mixed with a pipette. 500 µl 2 x HBS was added drop by drop.
After gentle shaking of the tube the reaction mix was incubated for 20 min at RT. The solution of 1 ml volume was added to the 10 cm dish drop by drop, while the dish was shaken gently. After 16 h the medium was changed. After a total of 48 h the medium from the cells, containing the viruses, was collected. The cell supernatant was centrifuged for 10
Methods 42
min at 2,500 rpm (Labofuge 400, Heraeus, Hanau, Germany) at RT to get rid of cell fragments. The supernatant was sterile filtered with 0.45 µm filters. The lentivirus containing medium was stored at 4 °C for further concentration or stored directly at -80 °C until use.
4.3.2 Concentration of lentiviruses with ultracentrifugation
4 ml of a 20 % sucrose solution was placed at the bottom of a hard shell centrifugation tube.
This solution was covered with maximally 26 ml of virus-containing medium. The centrifugation took place in an ultracentrifuge (LE-80K, Beckman-Coulter) at 26,000 rpm at 4
°C for 2 h in a SW32Ti Swing out rotor (Beckman-Coulter). The viruses concentrated as an often invisible pellet under the sucrose cushion. The supernatant was discarded and the pellet was resuspended in 100 µl sterile 1 % BSA/PBS per 10 ml virus medium used. The concentrate was stored at -80 °C.
4.3.3 Transduction of H6-22L cells with recombinant lentiviruses
For transduction 5 x 105 H6-22L cells were seeded on 6 cm plates. After 6 h the cells attached to the surface and were transduced with recombinant lentiviruses with a MOI of 10 or 15. The cells were incubated with the viruses for 72 h, before they were lysed and the
For transduction 5 x 105 H6-22L cells were seeded on 6 cm plates. After 6 h the cells attached to the surface and were transduced with recombinant lentiviruses with a MOI of 10 or 15. The cells were incubated with the viruses for 72 h, before they were lysed and the