Cell Signaling Lab Start: Monday, January 26, at 9:00 sharp (C6: Lab4/Cell lab) 1
Background PDGF-BB is a growth factor that bind and activates the tyrosine kinase receptor PDGFR. Activated PDGFR emits signals to many intracellular signaling pathways, for example Erk1/2 kinase cascade. In the adult human, PDGF signaling is important in wound healing when fibroblasts (and other cell types) should migrate to the wound and proliferate to cause healing of the wound. Over-activity of PDGF signaling have been found in various pathological conditions such as cancer (e.g. glioblastoma and gastrointestinal stromal tumors) but also in other diseases that are characterized by increased cell proliferation, for example atherosclerosis and fibrotic disease. The Erk1/2 pathway is activated by the receptor by recruiting the Grb2/Sos complex to the inner leaflet of the plasma membrane where it causes Ras activation. Subsequently the kinases Raf-1, Mek1/2 and Erk1/2 are activated downstream Ras. Activated Erk1/2 usually moves to the nucleus where it exerts its function by phosphorylating transcription factors that drive expression of genes necessary for cell-cycle progression. Overactivity of Erk1/2 cascade is a hallmark of most human tumors due to mutations in proteins within or upstream of the pathway. In this lab practical, you will culture NIH3T3 fibroblasts and treat them with PDGF-BB in the presence or absence of the Mek1/2 inhibitor U0126. You will observe, using immunofluorescence microscopy that upon PDGFBB stimulation Erk1/2 that initially is mostly cytoplasmic, translocates into the cell nucleus. You will see what effect inhibition of Mek1/2 has on this process. To be able to easily find cells you will also stain the actin cytoskeleton. Questions 1. How do trypsin and EDTA remove cells from the plastic? 2. What are the functions of fixation and permeabilisation of the cells? What do paraformaldehyde and Triton X-100 do? 3. What is the function of phalloidin? 4. What is the function of serum: a) during cell culture and b) during fluorescent staining? 4. What is the purpose of serum starvation before stimulation with the growth factor? 5. Why should you treat one set of the cells with DMSO? Key reagents needed Primary ab: p44/42 MAPK (Erk1/2) (L34F12) Mouse mab #4696 from Cell signaling Secondary ab: TRITC labeled anti-mouse antibody Actin staining: Phallodin-FITC Mek inhibitor: U0126 #9903 from Cell signaling PDGF-BB: Human Platelet-Derived Growth Factor BB (hpdgf-bb) #8912SC 2
Protocol Day 1: Harvest, seeding and starvation of the cells To be able to look at the cells in a fluorescence microscope you need to grow the cells on a Cover-slip. Today you are going to harvest the cells from the provided cell dishes using Trypsin/EDTA to detach then from dishes and then seed them on cover-slips. It is necessary to work sterile! (1) Carefully place cover-slips into 6-well plate using tweezers. You will need four wells. (2) Each group gets T-25 bottle with NIH3T3 cells. Take out the bottle from the incubator and look in the microscope. Aspirate the medium, rinse with 4 ml 1xPBS. Add 1-2 ml of Trypsin-EDTA solution to the bottle and incubate at room temperature for 5 min or until the cell detach (look in the microscope). Detach the cells completely by knocking the bottle carefully on the table. (3) Add 4 ml cell culture media DMEM_10 (DMEM + 10% FBS +antibiotics) and re-suspend the cells in the bottle. (4) Transfer the cell suspension to a 15 ml Falcon tube and centrifuge at 1000 rpm for 5 min. Aspirate the supernatant and carefully re-suspend the cell pellet (slowly pipette up-and-down a few times) in 4 ml DMEM_10 culture media (Rising step should remove Trypsin completely). Spin down again at 1000 rpm for 5 min to pellet cells. Aspirate the supernatant and carefully re-suspend the cell pellet again (by slowly pipetting upand-down a few times) in 9 ml DMEM_10 culture media. (5) To each well (containing cover-slip) add 2 ml of DMEM_10 cell culture media and 1 ml of cell suspension from the Falcon tube (from step 4). (6) Allow the cells to attach to cover-slip (3-4 hrs) in the Incubator. Then aspirate the cell culture media and replace it with 3 ml of starving DMEM_0.1 (DMEM + 0.1% FBS +antibiotics) and incubate overnight in the incubator (37C + 5% CO2). 3
Day 2: Inhibitor, growth factor treatment, staining Today you will stimulate the cells. 1. Add 6 ul of 5 mm U0126 already dissolved in DMSO to two of the wells to make 10 M final U0126 concentration and an equal volume DMSO to the other two wells. Mix carefully and incubate 1 hr in the incubator (37C / 5% CO2). 2. The cells in two of the wells (one treated with DMSO and the other with U0126) should be stimulated with 50 ng/ml final PDGF-BB concentration for exactly 10 min (10 +/- 1 min). Prepare ice box and ice cold PBS in advance (Look what you will do at the next step #3 too!). To stimulate the cells, add 15 l of 10 ng/ l PDGF-BB in DMEM_0.1 per well, mix carefully and incubate for 10 min in the incubator (37C / 5% CO2). 3. Take the plate from the incubator, quickly aspirate the medium from the plate, place the plate on ice and wash once with 3 ml ice cold 1xPBS. Be sure you use Pastor pipettes without cotton plugs! 4. Remove the PBS and fix the cells with 4% paraformaldehyde (PFA) in PBS by adding 1-2 ml PFA solution to each well. Incubate for 15 min at room temperature. 5. Aspirate the PFA and rinse the wells 3 times with 3 ml of 1xPBS. Before removing the final PBS prepare permeabilisation solution. 6. Remove final wash of PBS and incubate the cells with permeabilisation solution (10% FCS, 0.2% Triton-X100 in PBS) for 5 min at room temperature (add enough solution, about 1-2 ml, to cover the cover slips). 7. Aspirate permeabilisation solution and wash the wells 3 times with 3 ml 1xPBS (add PBS with a pipette and aspirate it using vacuum). 8. Block un-reacted PFA by adding 3 ml of 20 mm glycine in PBS (ph 7.4) per well and incubate for 1 hr at room temperature 9. Aspirate glycine solution and wash the wells 3 times with 3 ml 1xPBS. 10. Prepare staining solution: Take diluted primary Erk antibody (Prepared in advance:1:50 dilution of the stock in 5% FBS in PBS). Put a 15 ul drop of it onto para-film and put a cover slip onto it, the cell side of the slip facing the drop. Do not press onto the cover-slip! You may squash your cell! Incubate overnight at +4 C -+10 C (preferentially in a cold room). 4
Day3: Staining and evaluation Today you will look at the cells into the fluorescence microscope. You do not have to work sterile. You will draw how the cells look like and discuss the differences. Bring paper and pencil for the drawing. The drawings should be included in the lab report. 11. Put cover slips into the wells of a new plate with the cell side of the slip up and wash 6x in 1xPBS to remove the primary antibody from outside the cells. 12. Prepare secondary antibody solution: Dilute anti-mouse TRITC (gives red color, to stain Erk) conjugated antibody diluted 1:40 and phallodin-fitc to 20 g/ml (gives green color, to stain the actin cytoskeleton) in 5%FCS in PBS. Put a 15 ul drop of it onto para-film and put a cover slip onto it, the cell side of the slip facing the drop. Do not press onto the coverslip, you may squash your cell! Cover the tray with your reaction with aluminum foil and incubate for 1h at room temperature. 13. Wash 6x in 1xPBS and once with water. Do not remove the water before you are ready with microscope slides! 14. Put a drop (about 10 μl, cut tip for pipetting) of PVA/NBD onto a labelled slide, take out the coverslips with tweezers and put them carefully onto the slide upside-down, so that the cells end up in between coverslip and slide!!. Do not press onto the coverslip! Seal the coverslip with nail polish to avoid sliding the coverslip along slide surface. Now you are ready to look at the cells in the fluorescence microscope (only one group at a time - see separate schedule). Bring paper and pencil to draw your observations. The fluorophores are subject to bleaching. Avoid exposing the specimens to light unnecessarily, during microscopic observation or outside the microscope. 5
Cell culture work guidelines: No bags, coats etc. are allowed in the cell lab. You must arrange a locker before the start of the lab course where you can keep your clothes. Contact the BMC reception, corridor B7:1. Wear lab coat and gloves whenever working with cell culture. Bring your own lab coat! Lab coats are not allowed outside the lab areas. No food or drinks are allowed in the lab. Contact lenses are only permitted in combination with safety goggles. Please turn off your mobile phone while working in the lab. In cell culture, aseptic working technique must be observed to prevent environmental germs from contaminating the hood and incubator and infecting the culture. Before and after work in the sterile hood clean the working area with 70% ethanol. Spray everything with 70% ethanol before you place things into the hood; this includes the gloves you are wearing. Dishes with cells are kept in the incubator, in the sterile hood or under the microscope. Do not put them onto dirty lab benches or on the floor, as this will contaminate them and subsequently contaminate the sterile working areas with germs! Wash Lab bench with 70% EtOH. The lab assistants can relegate you from the lab if you do not follow the rules. Waste: Things that have been in contact with cell suspension belong into the boxes labelled in yellow with Riskavfall. Sharp and pointy things belong into the blue tagged boxes labelled Stickande och Skärande, while gloves and paper can be discarded in the normal waste bin (unless they have been in contact with cell suspension). 6
Writing the lab report: You should write one report for each lab group. Everyone has to contribute equally. The lab report describes your work and presents your results from the lab course. In order for your lab report to be easy to read and understood it must contain the following sections: Introduction In the introduction you should state the purpose of the practical. Give a brief background theory (e.g. on the cell cultures, etc.) and the techniques you are using. Materials and methods In materials and methods you describe, in your own words, how you performed the experiment. Include amounts, concentrations, incubation times etc. in the running text. You should be able to perform the experiment without additional information. Try to be as precise as possible but try to reduce this part to approximately half a page. Remember the most important parts of your lab report are the results and the discussion of them! Use past tense (in Swedish: imperfekt ). ) i.e. do not use words as I, he and we, when writing the materials and method section. Results Consist of two parts: (1) running text, which describes the observed results and (2) figures plus figure legends. Describe the results you obtained in a running text but do not interpret / analyse them in this section. Make the results easy to read e.g. by referring to tables and figures. The obtained results are shown in figure(s) and or table(s) in an understandable way (decide what is important in order to understand the figure). The figure legend should give enough information, so that the figure can be understood, without reading the entire report (what do you see? (e.g. which cell line), how did you treat your samples? (e.g. PDGF, include concentration and incubation time, visualisation method (e.g. staining), magnification of microscope, etc.). Include your drawings of the different cell lines or photographs (if taken from the web add the reference). Discussion Interpret the results, draw conclusions based on your results and biological facts. Include text book information in your discussion. Add references where necessary (e.g. quotation of text book, articles, web pages, etc.) Did you achieve the desired results? If not, give possible reasons why; sources of errors, what you would do to improve your experimental procedures etc. OBS!! If you got the expected results, stating "Everything worked as expected" is not discussing the results!!! The report must include your group number, the names and email addresses of the group members. The ready report should be sent as a Word file to mpavlov@xray.bmc.uu.se 7