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• 10:47, 7 March 2025 ESO wikiadmin talk contribs created page Immunofluorescence staining (Created page with "{| class="wikitable" |+ !Materials ! ! ! |- | |PBS++ (50 ml PBS; 25 μl 2.5M CaCl₂ ; 1M 50 μl MgCl₂) | | |- | |Permeabilization solution (PBS++; 10% FCS; 0.2% Saponin; alternatively 0.2% | | |- | |Triton X-100 may be used instead of saponin as a detergent) | | |- | |Blocking solution (PBS++; 10% FCS) | | |- | |First and second antibody | | |- | |Mounting medium | | |- | |Nail polish | | |} === Procedure === ---- * After fixation wash (3 x with PB...") Tag: Visual edit
• 10:45, 7 March 2025 ESO wikiadmin talk contribs created page Preparation of samples for confocal microscopy (Created page with "{| class="wikitable" |+ !Materials ! |- | |Coverslips (round, 12 mm) |- | |24-well plate |- | |4% Paraformaldehyde (PFA) in PBS or |- | |ice-cold methanol or acetone (-20°C) in porcelain well plate |} === Procedure === ---- Cells: * One day before seeding the cells: distribute cleaned and sterilized coverslips in a 24-well plate and coat with gelatine-, poly-L-lysine (10 µg/mL), or fibronectin (2 µg/mL) solution in PBS overnight (o/n) at 4°C * Aspirate coating so...") Tag: Visual edit
• 10:43, 7 March 2025 ESO wikiadmin talk contribs created page Direct labelling of antibodies with fluorescent dyes (Created page with "{| class="wikitable" |+ !Materials ! |- | |Pacific Blue succinimidyl ester (absMAX= 416nm; emMAX= 451nm) |- | |5-(6)-carboxyfluorescein succinimidyl ester (FAM, SE) (absMAX= 494nm; emMAX= 518nm) |- | |5-(6)-carboxytetramethylrhodamine succinimidyl ester (TAMRA, SE) (absMAX= 555nm; emMAX= 580nm) |- | |AlexaFluor-647 succinimidyl ester (absMAX= 650nm; emMAX= 665nm) 1 x phosphate-buffered saline (PBS) |- | |1M sodium bicarbonate (pH 8.5) |- | |STOP buffer (prepare fresh): 1...") Tag: Visual edit
• 14:44, 27 February 2025 ESO wikiadmin talk contribs created page Dot Blot (Created page with "{| class="wikitable" |+ !Materials ! |- | |Nitrocellulose membrane / Whatman paper |- | |TBS-T |- | |Ponceau S |- | |Blocking Solution |- | |First and Second Antibody |- | |ECL solution and H₂O₂ |} === Procedure === ---- * Whatman paper and membrane have to be wet  first whatman on dot blot, followed by membrane. Top of dot blot holder has to be firmly secured with screws on the lower part! * Apply 50 µl of probe with equivalent amount of prot...") Tag: Visual edit
• 14:40, 27 February 2025 ESO wikiadmin talk contribs created page Western Blot: stripping of the membrane (Created page with "{| class="wikitable" |+ !Materials ! |- |Mild Stripping Buffer |15 g Glycin, 1 g SDS, 10 ml Tween20 in 1L ddH₂O pH2.2 |- |Harsh Stripping Buffer |40 ml Stacking gel buffer (0,5M Tirs/HCl, pH6,8) 10 ml SDS 20%, 200 ml ddH₂O |- | |1X PBS |- | |TBST |} == Procedure mild stripping == * Remove ECL from membrane by washing membrane briefly with ddH₂O * Wash membrane 2x in Mild stripping buffer for 10 min * Wash membrane 2x in PBS for 10 min *...") Tag: Visual edit
• 14:29, 27 February 2025 ESO wikiadmin talk contribs created page Western Blot: probing of the membrane (Created page with "{| class="wikitable" |+ !Materials ! |- |Staining Solution |250 mL isopropanol; 100 mL acetic acid; 650 mL H₂O; 0.03% Coomassie |- |Destaining Solution |10% (v/v) isopropanol, 10% (v/v) acetic acid |- |Blocking Solution |TBS-T; 2% BSA; 0.05% NaN₃ |- |TBS-Tween |200 mL 10x TBS; 10 mL Tween; ad 2 L H₂O |- | |ECL-Solution |- | |H₂O₂ (30%) |} === Procedure === ---- * Upon completed transfer of the proteins to the PVDF...") Tag: Visual edit
• 14:26, 27 February 2025 ESO wikiadmin talk contribs created page Western Blot: semi-dry blot (Created page with "{| class="wikitable" |+ !Materials ! |- |5X Anode buffer |15 g Tris Base, pH to 10.4, final volume 1l |- |5X Catode buffer |15 g Tris Base, 26.25 g 6-amino-N-caproic acid (6-Amino-hexansäure), pH 9.4; final volume 1 l |} === Procedure === ---- * Anode Buffer preparation: 20 ml 5x Anode buffer, 10 ml MeOH, fill up to 100 ml with ddH2O * Cathode Buffer preparation: 20 ml 5x Cathode buffer, 10 ml MeOH, fill up to 100 ml with ddH2O * The PVDF membrane is labeled with a pe...") Tag: Visual edit
• 14:23, 27 February 2025 ESO wikiadmin talk contribs created page Western Blot: wet blot (Created page with "{| class="wikitable" |+ !Materials ! |- |Western Transfer Buffer |dissolve 6.0 g Tris Base; 28.8 g glycine in 1 L dH₂O add 430 mL methanol and degas for 30 min while stirring after degasing, add 10 mL 20% SDS and fill up to 2 L with H₂O |- |Blocking Solution |TBS-T; 2% BSA; 0.05% NaN₃ |- |TBS-Tween |200 mL 10x TBS; 10 mL Tween; ad 2 L H₂O |- | |First and second Antobody |- | |ECL-Medium |- | |H₂O₂ (30%) |-...") Tag: Visual edit
• 14:21, 27 February 2025 ESO wikiadmin talk contribs created page File:Schema Sandwich WB.jpg
• 14:21, 27 February 2025 ESO wikiadmin talk contribs uploaded File:Schema Sandwich WB.jpg
• 14:15, 27 February 2025 ESO wikiadmin talk contribs created page Coomassie Blue staining of gels (Created page with "{| class="wikitable" |+ !Materials ! |- |Staining Solution |25% (v/v) isopropanol 10% (v/v) acetic acid 0.03% (w/v) Coomassie Brillant Blue R250 |- |Destaining Sol. |500 mL methanol; 500 mL dH₂O 100 mL acetic acid |} === Procedure === ----The gel is heated in Coomassie staining solution briefly in the microwave and incubated for 30 min on a shaker. After this time it is incubated in destaining solution, so that the blue protein bands are visible against a cle...") Tag: Visual edit
• 14:13, 27 February 2025 ESO wikiadmin talk contribs created page SDS-PAGE (Created page with "{| class="wikitable" |+ !Materials ! |- |Separating Gel (12,5%): |3.1 mL Polyacrylamide (40%); 4.3 mL dH₂O; 2.5 mL Tris (1.5 M; pH 8.8) ''Degas!'' 50 μL Sodium dodecyl sulfate (SDS; 20%); 30 μL Ammonium Persulfate (APS; 10%); 15 μL TEMED |- |Stacking Gel (5%): |1.25 mL Polyacrylamide (40%); 6.15 mL dH₂O; 2.5 mL Tris (0,5 M; pH 6,8) ''Degas!'' 50 μL Sodium dodecyl sulfate (SDS; 20%); 30 μL Ammonium Persulfate (APS; 10%); 15 μL TEMED |- |Marker...") Tag: Visual edit
• 14:10, 27 February 2025 ESO wikiadmin talk contribs created page Whole cell lysates (WCLs) of eukaryotic cells (Created page with "{| class="wikitable" |+ !Materials ! |- | |PBS 1X |- | |RIPA buffer |- | |Sepharose Beads |} === Procedure === ----All following steps are performed in the cold room: * Aspirate cell culture medium with a pasteur pipette * Rince attaching cells carefully with cold PBS * Add 1 mL RIPA buffer per 10 cm dish or 0.5 mL per 6 cm dish and incubate for approx. 2 min at 4°C * Carefully detach the cells with a cell scraper and transfer the lysate in a fresh tube * Mechanical s...") Tag: Visual edit
• 13:53, 27 February 2025 ESO wikiadmin talk contribs created page Measurement of endocytosis via reversible surface biotinylation (Created page with "{| class="wikitable" |+ !Materials ! |- |Biotinylation buffer: |PBS++ (pH 7.4) + 400 µg/ml Sulfo-NHS-SS- Biotin (freshly added) |- |Quenching buffer: |PBS++ (pH 7.4) + 100 mM Glycin |- |Stripping buffer: |50 mM Tris (pH 8.6), 100 mM NaCl, freshly added: 0.2 % BSA, 100 mM MESNa |- |Serum starvation medium: |DMEM + 0.5 % calf serum (CS) |} === Procedure === ---- * 2 x 105 cells in 6 cm dishes, serum starvation over night * Wash 3x with cold PBS++ at 4°C * Incubatio...") Tag: Visual edit
• 13:50, 27 February 2025 ESO wikiadmin talk contribs created page Preparation of deterg.-resist. membrane microdomains (Sucrose Gradient) (Created page with "{| class="wikitable" |+ |'''Materials''' | colspan="3" | |- |Homogenisation buffer |50 mM Tris pH 7.4 |[0.5M] |▶ 10 ml |- | |2 mM MgCl₂ 8 % sucrose |[1M] |▶ 200 µl ▶8 g |- | |ddH₂O | |▶ 89.8 ml |- |TNE buffer | colspan="2" |20 mM TrisHCL pH8 [1M] |▶ 2 ml |- | | colspan="2" |130 mM NaCl [5M] |▶ 2.6 ml |- | | colspan="2" |5 mM EDTA [0.5M] |▶ 1 ml |- | | colspan="2" |10 µg/ml Aprotinin [5mg/ml] |▶ 200 µl |- | | colspan="2" |10 µg/...") Tag: Visual edit
• 13:45, 27 February 2025 ESO wikiadmin talk contribs created page Measurement of lipid raft localization via flow cytometry (Created page with "{| class="wikitable" |+ !Materials ! |- | |PBS (pH 7.4) Trypsin/EDTA Triton-X100 Serum starvation medium: 0.5% calf serum (CS) FACS buffer: 46.5 ml PBS + 2.5 ml FCS (h.i) + 1 ml 5% sodium azide |} === Procedure === ---- * Add cells in poly-L-lysin coated dishes * Optionally: Serum starvation of cells over night, 37 °C * Positive control: fluorescently labelled choleratoxin for staining of Gm1 * Negative controls: staining of transferrin receptor, untransfected cells...") Tag: Visual edit
• 13:41, 27 February 2025 ESO wikiadmin talk contribs created page SEAP Reporter Assay (Created page with "{| class="wikitable" |+ !Materials ! |- | |Eppi centrifuge at RT Heat block @65°C Substrate buffer @+4°C pNPP powder Calf Intestine Alkaline Phosphatase (CIAP) [stock: 1μL/mL, 1:1000 dilution) Multichannel pipette and plastic box for solution Spectrophotometer (VarioSkant) |- |Assay buffer (100ml) |100 mM Glycin pH 10.4 [10 M] ▶ 1 mL 1 mM ZnCl2 [1 M] ▶ 400 μL 1 mM MgCl2 [1 M] ▶ 400 μL Adjust pH with NaOH to pH 10.4 |} === Procedure === ---- * Transfer  ...") Tag: Visual edit
• 13:38, 27 February 2025 ESO wikiadmin talk contribs created page Luciferase Reporter Assay (Created page with "{| class="wikitable" |+ !Materials ! ! ! |- | |Ice bath for buffers, Luciferin and lysates PBS rubber policeman (cold room) and Eppis for lysates, Eppi centrifuge at RT Lysis buffer (1M DTT → 2 µl per ml) (fridge 4°C) Assay buffer (1M DTT → 10 µl per 10 ml; 200 mM ATP → 100ul per 10 ml) (fridge 4°C) Luciferin aliquots (-20°C freezer) Multipipette and plastic box for solution (cell culture room) | | |- |Assay buffer (100ml): |25 mM Tris pH 7.8 [1 M] → 2.5 ml...") Tag: Visual edit
• 13:35, 27 February 2025 ESO wikiadmin talk contribs created page Granulocyte oxidative burst (Created page with "{| class="wikitable" |+ !Materials !and Preparations |- | |2 days before experiment, streak out gonococci and select the colony phenotype one day before experiment. |- | |Chemiluminescense buffer (CL-Buffer): 8 g NaCl; 0.2 g KCl; 0.62 g KH₂PO₄; 1.14 g Na₂HPO₄; 2.5 ml 40% glucose; 50 mg BSA; solve in 800 ml bidest. H₂O; adjust pH to 7.2 and fill it up to 1 liter; steril filter and store at 4°C. |- | |0.2 mg/ml PMA (P...") Tag: Visual edit
• 13:31, 27 February 2025 ESO wikiadmin talk contribs created page StrepTactin-Pulldown (Created page with "{| class="wikitable" |+ !Materials ! |- |Recombinant protein |Bait protein TwinStrepTagII tagged protein, target proteins (HisSUMO or GST tagged protein of interest) |- |Pull down buffer |50 mM Tris/Cl pH 8, 150 mM NaCl, 10% Glycerol, 0.05% Tween. Optional: 10 µM ZnCl₂ in case of Zinc- finger proteins or 2 mM MgCl₂, CaCl₂ or MnCl₂ in case of metalloproteins) |- |Elution buffer EXT |50 mM Tris/Cl pH 8, 150 mM NaCl, 50 mM Biotin...") Tag: Visual edit
• 13:27, 27 February 2025 ESO wikiadmin talk contribs created page Rac pulldown assay (with GST-PBD beads) (Created page with "{| class="wikitable" |+ !Materials ! !''add fresh'' |- |Lysis Buffer |Tris pH 7.5, 50 mM, NaCl 150 mM, MgCl2 10 mM, NP40 1%, Glycerol 10% | rowspan="2" |DTT 1 mM, Leupeptin, Aprotinin, PMSF |- |Wash Buffer |Tris pH 7.5, 25 mM, NaCl 40 mM, MgCl₂ 30 mM, NP40 1% |- | |Glutathione-Sepharose beads with bound recombinant GST-PAK-binding-domain (GST-PBD) | |} === Procedure === ---- * Prepare lysis and wash buffer and keep on ice. Thaw beads (-80°C) on ice. * Stimu...") Tag: Visual edit
• 13:16, 27 February 2025 ESO wikiadmin talk contribs created page GST-Pulldown (Created page with "{| class="wikitable" |+ !Materials ! |- |GST-Lysis Buffer: |50 mM Tris-HCl (pH 8), 150 mM NaCl, 10% glycerol, 0.05% sodium deoxycholate, 1% Triton- X, 10 µM ZnCl2, 50mM NaF (add protease and phosphatase inhibitors freshly on the day of the experiment) |- |GST-Pulldown/wash buffer: |50 mM Tris-HCl (pH 8), 150 mM NaCl, 10% glycerol, 0.05% sodium deoxycholate, 1% Triton- X, 10 µM ZnCl2, 50mM NaF (add 1mM DTT freshly on the day of the experiment) |- | |Purified recombinant...") Tag: Visual edit
• 13:12, 27 February 2025 ESO wikiadmin talk contribs created page Fibronectin-Binding Assay (Created page with "{| class="wikitable" |+ !Materials ! ! ! |- | |humanes Serum (500µl/Probe) | | |- | |gekoppelte Latex Beads | | |- | |α-Fn Antikörper (HFN7.1) | | |- | |Zweitantikörper (α-MCy2) | | |} === '''Durchführung''' === ----Von den zu untersuchenden Beads werden 30-60µl mit 500µl humanem Serum gemischt und 2h bei RT auf dem Rotator inkubiert. Anschliessend werden die Proben zweimal mit PBS gewaschen und mit □-Fn Antikörper versetzt (Zellkulturüberstand unverdünnt)....") Tag: Visual edit
• 13:11, 27 February 2025 ESO wikiadmin talk contribs created page Covalent coupling of proteins to latex beads (Created page with "{| class="wikitable" |+ !Materials ! ! ! |- |Borate buffer |0.2 M of boric acid with 1 M NaOH, adjusted to a pH value of 8.5 | | |- |2% Carbodiimide solution |2% 1-(3-Dimethylaminopropy)-3-ethyl-carbodiimide hydrochloride are dissolved in MES buffer. Prepare only 15 minutes before use | | |- |Carbonate buffer |0.1 M Na₂CO₃ are added to 0.1 M NaHCO₃ until a pH value of 9.6 is reached | | |- |MES buffer |0.1 M MES in water, pH value of 5.2-...") Tag: Visual edit
• 13:08, 27 February 2025 ESO wikiadmin talk contribs created page In vitro Tyrosine Kinase Activity Assay (Created page with "{| class="wikitable" |+ !Materials ! |- | |Kinase buffer (125 mM NaCl; 48 mM MgCl₂; 50 mM HEPES (pH 7.5) |- | |4x SDS Buffer |- | |1 mM ATP |- | |Purified Kinase (e.g. Src or FAK) |- | |Kinase substrate (e.g. CEACAM3-cytoplasmic domain or FAK substrate) |} === Procedure === ---- * 0.2 μg/ml FAK-substrate or 25 ng/μl TwinStrep-Ceacam3-cytoplasmic domains * 0.2 μg/ml hFAK KD (recombinant human FAK kinase domain) or 10 ng/μl Src * with or without (control)...") Tag: Visual edit
• 13:06, 27 February 2025 ESO wikiadmin talk contribs created page Protein Phosphatase Assay (Created page with "{| class="wikitable" |+ !Materials ! |- | |black flat-bottom 384-well plates (Greiner Bio-One, Germany) |- | |phosphatase buffer (50 mM TrisHCl, pH = 8.0 at 25°C, 200 mM NaCl, 20 mM MnCl2, 2 mM TCEP, 0.01% Tween). TCEP and Mn²⁺ are added freshly; 1L bottle is sterile filtered and stored at 4°C. |- | |4-Methylumbelliferyl phosphate (4-MUP) (Sigma Aldrich, Germany) or DiFMUP (ABCR GmbH) is dissolved in DMSO (10 mM stock solution) and stored at -50°C |- | |rec...") Tag: Visual edit
• 13:05, 27 February 2025 ESO wikiadmin talk contribs created page File:Example384wellplate.png
• 13:05, 27 February 2025 ESO wikiadmin talk contribs uploaded File:Example384wellplate.png
• 12:43, 27 February 2025 ESO wikiadmin talk contribs created page Determination of Integrin Activity (ELISA-based) (Created page with "{| class="wikitable" |+ !Materials ! ! ! |- |'''Wash solution''' |1x PBS mit .0.0 5% BSA | | |- |'''Block solution''' |2% BSA in 1 xPBS | | |- |'''Permeabilization solution''' |0.1% Triton X-100 (0.1% in PBS) | | |- |'''Buffer for antibody''' |0.1% BSA in x PBS | | |- |'''Reaction buffer''' |10 ml Lösung '''B''' + 0.5 ml Lösung '''A''' | | |- |'''Solution A''' |120 mg Tetramethylbenzidin in 5 ml Aceton + 45ml EtOH add 100µl H₂O₂ | | |- |'''Soluti...") Tag: Visual edit
• 12:36, 27 February 2025 ESO wikiadmin talk contribs created page Cell Adherence assay (Created page with "'''Materials''' {| class="wikitable" |+ !Materials ! |- | |96 well plates; 15 ml und 30 ml Falcon-tubes |- | |coating solution for wells e.g. collagen type 1 (25 µg/ml) |- | |Soybean Trypsin/Inhibitor (12.5 mg/50 ml) in Suspensionen medium- sterile |- | |Crystal violet (250 mg/ 5 ml 96% Ethanol stock solution) |- | |Suspension medium (DMEM +0,25%BSA) sterile |- | |diluted Crystal violet 1:100 in 0.1M Borat buffer pH: 9.2 (freshly prepared!) |- | |4% PFA (Paraformaldehyd...") Tag: Visual edit
• 12:28, 27 February 2025 ESO wikiadmin talk contribs created page Cell viability (MTT Assay) (Created page with "===== Solutions ===== MTT-Solution: 5mg/mL (≙12mM) (toxic and mutagenic) solved in PBS, sterile filtered with 0,2 µM filter Formazan solving solution: isopropanol === Procedure === ----Coat plate before usage For 96 well plates: use 1 - 2x104 cells in 100 µL medium per well and treat the cells according to your experimental setup. The cell number above was optimized for 1 day treatment; for longer incubation periods use fewer cells accordingly. * After treatme...") Tag: Visual edit
• 18:55, 15 February 2025 ESO wikiadmin talk contribs created page Production of soluble CEACAM domains in human cells (Created page with "{| class="wikitable" |+ !Materials ! ! ! |- | |Plasmid DNA | | |- | |DMEM |Cell culture medium with L-glutamine and 4.5 g/l glucose (PAA Laboratories) | |- | |10% CS |calf serum (PAA Laboratories) | |- | |2xHBS |16.4 g NaCl, 11.9 g HEPES, 0.21 g Na₂HPO4, solve in 1 l ddH₂O, pH 7, sterile filtration | |- | |CaCl₂ solution |2.5 M CaCl₂ | |- | |OptiMEM |Serum-reduced medium with L-glutamine and HEPES (Gibco) | |} === Procedure =...") Tag: Visual edit
• 18:52, 15 February 2025 ESO wikiadmin talk contribs created page Concentration of lentiviral particles via ultracentrifugation (Created page with "{| class="wikitable" |+ !Materials ! ! ! |- | |Virus containing supernatant | | |- | |20% sucrose in PBS | | |- | |1% BSA in PBS | | |} === Procedure === ---- # 4 ml of 20% sucrose solution are added to an ultracentrifuge tube and overlayed with 26 ml of filtrated virus containing supernatant # the tubes are placed in aerosol tight swing buckets # centrifugation is carried at 20.000 rpm , 4°C for 2h15min (Rotor: SW32Ti) # after discarding the supernatant the pellet is...") Tag: Visual edit
• 18:50, 15 February 2025 ESO wikiadmin talk contribs created page Viral transduction of mammalian cells via spinfection (Created page with "{| class="wikitable" |+ !Materials ! |- | |Virus containing supernatant |- | |1000x Polybrene (Hexadimethrine bromide) in PBS (8mg/ml) 1*106 suspension cells or 1*105 adherent cells |- | |24 well plate (for suspension cells) 6 well plate (for adherent cells) |} === Procedure === ---- # 1 ml medium containing 1*105 or1*106 cells are added into 6 or 24 well plate. # Add 2 µl 1000x Polybrene # Add 1ml of virus supernatant or a corresponding dilution of concentrated virus...") Tag: Visual edit
• 18:48, 15 February 2025 ESO wikiadmin talk contribs created page Transduction and titration of lentiviral supernatant (Created page with "{| class="wikitable" |+ !Materials ! |- | |DMEM + 10% CS |- | |Puromycin (stock solution 5 mg/ml) |} === Procedure === # 1x10⁴ 293T cells are seeded in a 24-well plate ~12 h before transduction (5 wells) # 1µl, 2µl or 5µl, respectively, of the virus-concentrate are added on top of the cells, 2 wells remain untreated # incubation over night at 37°C and 5% CO₂ # 24 h after transduction the medium is replaced by 1 ml fresh medium containing 10% C...") Tag: Visual edit
• 18:46, 15 February 2025 ESO wikiadmin talk contribs created page Production of lentiviral particles (Created page with "{| class="wikitable" |+ !Material ! |- | |293T cells |- | |2x HBS-buffer |- | |2.5 M CaCl₂ |- | |25 mM Chloroquin |- | |Lentiviral vectors |} === Procedure === # A confluent plate of 293T cells is splitted 1:5 one day before transfection # The transfection reaction contains: 500 µl ddH₂O, 6.5 µg pLKO.1 (with gene or shRNA of interest), 3.5 µg pMD2.G (plasmid #1256) and 5 µg psPAX2 (plasmid #1257) # Add 500 µl 2x HBS-buffer # While vortexing t...") Tag: Visual edit
• 18:12, 15 February 2025 ESO wikiadmin talk contribs created page Infection of granulocytes (Created page with "=== Procedure === 1x10⁶ granulocytes are infected with bacteria (MOI 20) for 15 min at 37°C under rotation. Phagocytosis is stopped by the addition of 500 µl of ice cold PBS. Samples are centrifuged at 4°C for 5 min at 200 g and washed once with PBS. After centrifugation samples are taken up in PBS or SDS-buffer.") Tag: Visual edit
• 18:10, 15 February 2025 ESO wikiadmin talk contribs created page Granulocyte FACS phagocytosis assay (Created page with "{| class="wikitable" |+ !Material ! ! ! |- | |Freshly isolated granulocytes | | |- | |Phagocytosis buffer (PBS, 0.9 mM CaCl₂; 0.5 mM MgCl₂; 5 mM glucose; 1% heat inactivated FCS) | | |- | |5-(6)-carboxyfluorescein succinimidyl ester (FAM SE; CFSE, Fluorescein, FITC) | | |- | |Trypan Blue solution | | |} === Preparations === Two days before experiment: streak out gonococci on selective agar plates and select colony morphology one day before infectio...") Tag: Visual edit
• 18:06, 15 February 2025 ESO wikiadmin talk contribs created page Isolation of human granulocytes (Created page with "{| class="wikitable" !Materials ! |- | |Biocoll |- | |3.8% citrate solution PBS |- | |PVA 7200 polyvinyl alcohol 2x PBS, pH 7.4 |- | |Bidest H₂O |- | |Phagocyte buffer (PBS, 0.9 mM CaCl₂; 0.5 mM MgCl₂; 5 mM glucose; 1% h.i FCS) |} === Preparations === PVA1% PVA 7200 + 0.9% NaCl * Add 5 g PVA to 500 ml 0.9 % NaCl solution * Solve with stirring on a heating plate * Autoclave * Cool under stirring * Store on shelf, protected from light =...") Tag: Visual edit
• 18:00, 15 February 2025 ESO wikiadmin talk contribs created page SiRNA transfection with INTERFERin (Created page with "{| class="wikitable" |+ !'''Materials''' |- |siRNA |- |OptiMEM (serum-reduced medium) |- |INTERFERin |} === Procedure === Cells are seeded in 10 cm cell culture plates one day before transfection. 400 µl serum-reduced OptiMEM-medium and 10 µl (20µM) siRNA are mixed for 10 sec with 15 µl INTERFERin and incubated for 10-30 min at room temperature. The cell medium is replaced by fresh cell medium and siRNA preparation is added drop-wise to the cells and incubated for...") Tag: Visual edit
• 17:42, 15 February 2025 ESO wikiadmin talk contribs created page Transfection of fibroblasts (Created page with "=== Materials === Plasmid DNA OptiMEM Lipofectamin 3000 or jetPrime === Procedure === The day before transfection: seed fibroblasts on gelatine coated 24 or 6-well plate so that they are approx. 70% confluent at time of transfection (for NIH3T3 cells 2.5x10^4 for 24 well, 1.5x10^5 for 6 well plate) Change medium to remove and dead cells and debris Which transfection reagent works best for your cells has to be determined empirically: ==== '''JetPrime Protocol''' for...") Tag: Visual edit
• 16:31, 15 February 2025 ESO wikiadmin talk contribs created page Transfection of HEK293T cells (Created page with "{| class="wikitable" !Materials ! |- | |Plasmid DNA |- | |2x HBS- buffer, pH 7.05 (correct pH is extremely critical !!) |- | |2.5 M CaCl2 (sterile) |- | |25 mM chloroquin (sterile) |} '''Procedure''' * 1 day before transfection: cells split 1:3 up to 1:5 in 10cm dishes * Next day: Set up for each 10cm dish: 500 μl ddH2O (sterile) in 14 ml PP Greiner tube * Add 5 μg (or adapted amounts) of plasmid DNA * Add 500 μl 2x HBS buffer, mix sample * Add drop-wise and duri...") Tag: Visual edit
• 16:22, 15 February 2025 ESO wikiadmin talk contribs created page Cultivation of HEK293T cells (Created page with "'''Materials''' Medium: DMEM + 10 % calf serum (CS), PBS, Trypsin / EDTA '''Procedure:''' * Remove growth medium with sterile pasteur pipette from dishes * Wash 1x carefully with PBS, remove PBS * 1 ml Trypsin/EDTA on cells, short incubation (max 2 min) * If cells are detached, stop activity of trypsin with addition of 4 ml medium * Transfer cell solution in falcon tube, centrifugation 800rpm, 3 min, room temperature * Remove medium/ trypsin mixture, add new medium...") Tag: Visual edit
• 16:07, 15 February 2025 ESO wikiadmin talk contribs created page Rezepte (Created page with "= Zusammenfassung aller Rezepte = == Antibiotika-Lösungen == === Ampicillin (100 mg/ml) === '''Menge:''' 20 ml * 2.0 g Ampicillin * Dest. H2O ''Zusatzinfo:'' In Wasser lösen, Endvolumen 20 ml, steril filtrieren (0,2 µm), aliquotieren und bei -20 °C lagern. === Chloramphenicol (30 mg/ml) === '''Menge:''' 20 ml * 0.6 g Chloramphenicol * 96% EtOH ''Zusatzinfo:'' In Ethanol lösen, steril filtrieren, aliquotieren und bei -20 °C lagern. === Kanamycin (50 mg/ml) =...") Tag: Visual edit
• 00:12, 15 February 2025 ESO wikiadmin talk contribs created page MediaWiki:Sidebar (Created page with " * navigation ** mainpage|mainpage-description ** AG_Hauck_-_LabWiki-url|Startseite ** recentchanges-url|recentchanges ** randompage-url|randompage ** helppage|help-mediawiki * SEARCH * TOOLBOX * LANGUAGES")
• 17:06, 14 February 2025 ESO wikiadmin talk contribs created page Template:Box (Created page with "<div class="my-box"> <div class="my-box-title">{{{Titel}}}</div> <div class="my-box-content"> * {{{Inhalt|}}} </div> </div>")
• 17:05, 14 February 2025 ESO wikiadmin talk contribs created page Lab Etiquette (Created page with "{{Box |Titel=Themenübersicht |Inhalt= * Punkt 1 * Punkt 2 * Punkt 3 }}")
• 17:04, 14 February 2025 ESO wikiadmin talk contribs created page MediaWiki:Common.css (CSS Code für Box)
• 17:03, 14 February 2025 ESO wikiadmin talk contribs created page Vorlage:Box (Neue Box-Vorlage erstellt)
• 10:20, 14 February 2025 ESO wikiadmin talk contribs created page File:Zentri.jpeg (Bild einer geöffneten Zentrifuge)