1 Lab 3 Cell Biology 2015, Kristina Ruuth Characterisation of novel anticancer drugs PART 2Most currently used anticancer drugs work by targeting the cytoskeleton, cell cycle progression or the apoptotic machinery.Tumor resistance to anti-cancer drugsRef Per Holmfeldt/Mikael Sellin, Umeå University
2 Lab3: Each group will test 7 substanses from a chemical library plus 1 solvent control on Jurkat cellsCell viabilty:1. Tryptan blue exclusion after 24 and 48 h.2. Metabolic activity: Cell mediated reduction of resazurin after 24 h.Giemsa stain.FACS analysis:Cells double stained with immuno fluorescence technique for micro tubules and PI for cell cycle status.Dipak M et al Nature Comm 2013
3 Cell viabilty:Trypan blue exclusion: Counting of trypan blue stained cells in a burker chamber.Count all samples in triplicate at 2 time points , h and 48h after start of treatment.Triplicate: Count viable cells in at least 3 “A-squares” calculate average . Repeat this twice for each treatment and the solvent control.Cell conc 0.5x106 cell/mlDiluted 1:2 will result in approx 25 cells/A-Square with large fluctuations.Hints: Dilute 4:5 , eg 200uL cells + 50 uL Trypanblue .Take out samples for counting and place the tubes on ice. Storage on ice for a couple of hours will not affect cell viability.
4 Metabolic activity, Proliferation assay. Cell ViabilityMetabolic activity, Proliferation assay.Many assays: MTT, WST, 3H-thymidine, Brdu incorporation, Resazurin.590nm530nmResazurin (Alamar Blue):Medium +resazurinUntreated cells +Analyse all samples, in triplicate wells. Seed 150 uL/well, according to template, 96-well plate, 0.5x106 cells/mLIncubate 24h, add 50 uL/well of resazurin (final conc in wells 55 uM) incubate 2-3h 37°. Read generated fluorescence in Tecan M200 multiplate reader . Excitation set to 530 and emission set to 590 nm. Resazurin stock solution 4.4 mM.The reader is booked for 1hr at 3 occasions onTuesday 10/3:12:00-13:00, 14:00-15:00, 16:00-17:00
5 3. Giemsa/May Grunewald stain. Consider results from the two viability assays “trypan blue exclusion” and “resazurin assay.” Which samples from the chemical library affected growth?Choose 4 samples together with the solvent control and spin out cells on slides and stain cells with May Grunewald/Giemsa according to instructions given in Lab 1.After staining look through the slides and search for a phenotype for that specific drug.Estimate ratio “phenotypic cells”/normal cells among cells.
6 Methods to distinguish out stages in the cell cycle: G0 from G1.Ki-67 is not expressed in G0 cellsG0/G1SG2/MRHas cells passed the G1 restriction point.Western blot of cell lysates with Rb-antibodiesRbpRbactin
7 How to resolve different stages within G2/M G0/G1SG2/M
8 FACS analysis of micro-tubules Jurkat cellsRef Dipak M et al Nature Comm 2013μ-tubulesControl DrugIntact cellsPerforation of plasma membrane inpresence of saponin and taxol; Unpolymerised tubulins will diffuse out of the cellRef: Per Holmfeldt, Umeå universityAfter fixation incubate cells with anti-tubulin antibodies followed by incubation with FITC-labelled secondary antibodies.
9 Staining of microtubules for FACS (copied from “-MG lab methods 2011-“)Spin down cells per FACS tubes for 2 min, 1100rpm (250xg).Aspirate the supernatant and resuspend the pelleted cells gently using the shaking table.Extraction of cells; add 400µl 37 °C MT-STAB and incubate at 37 °C for 4 minutes.Fixation of cells; add 400 µl 37°C MT-FIX and incubate at37 °C for 12 minutes.Spin down the cells, 3 min, 1100rpm.Aspirate the supernatant and resuspend the cells by shaking.Add 1 ml BLOCK and incubate at 37 °C for 10 minutes.Spin down the cells, 5 min, 1100rpm.Add 150 µl primary antibody (anti--tubulin T5168 B dil 1:250 in BLOCK). NOTE! Include a tube with no addition of primary antibody to define the background.Incubate at 37 °C for 60 minutes while shaking.Add 1 ml WASH.Add 100 µl secondary antibody (Rabbit-anti-mouse (FITC) DAKO F0313 dilute 1:20 in WASH).Incubate at 37 °C for 30 minutes while shaking.Add 500µl PI SOL and keep samples in refrigerator until analysis.Solutions:MT-STABPEM buffer, pH 6.90.1 % Saponin (stock 10%)10 µg/ml RNase (stock 5mg/ml)50 nM Taxol (stock 1,1mM)MT-FIX~50 % PEM buffer, pH 6.9~50 % PFA in H2O (from 8% stock)0.1 % Saponin (add 1/100 of a 10 % stock, which may be stored ~1 week)BLOCK90 % PBSA / 10 % FCS / 0,1 % SaponinWASHPBSA / 0,1 % SaponinPI SOL.PBSA10µg/ml Propidium Iodide0,1% Triton X-100 (add 1/100 of a 10 % stock)10µg/ml RNase
10 micro-tubules är very britle Extraction of“free” tubulinAdd 400 μLMT-STAB 37°inc 37° 4 min.Fixation of cellsAdd 400 μL MT-FIX 37° inc 37° 12 minLoosen pellet by gently shaking on shakerboard500 μLcellsAspiratemediumCentrifuge1100 rpm3 minCentrifuge1100 rpm2minAdd 150 μL Mouse anti-α–tubulinAntibody and inc60min 37° in shakingwaterbath.Blocking of unspecificprotein binding sites.Add 1ml BLOCK andinc 10min 37°Add 1 mLWASHCentrifuge1100 rpm5 minAspirate supand shake gentlyAspirate supand shake gentlyCentrifuge1100 rpm5 minAdd 100 μL Secondary ab:FITC Rabbit α mouse IgG, inc 30min 37° in shaking waterbathAdd 0.5ml PI-soution.Store cells in fridge until FACS analysisAspirate supand shake gentlyAdd 1 mLWASHCentrifuge1100 rpm5 minAspirate supand shake gently
11 Structural effects on microtubules After FACS analysis of cells stained for u-tubulesAdjust cell cons to approx 0.5x106/mL and resuspend the cells. Spinn out 0.1x106 cells onto slides. Mount with “anti-fade” mounting medium and cover with coverslip.Let dry until next day an inspect the micro-tubule structure with a fluorescence microscope. Describe the structrual changes, count the phenotypic cells vs normal cells.Microscope slides with wellsan alternative to cytospinningAdjust cellconc to approx 0.5x106/mL.Label wells on slide, 12 wells/slide and put it into ”humified chamber”. Pipet 25 μL/well of resuspended stained cells in PI-sol. Allow cells to sediment onto glass for 2h in the dark. Remove excess of solution with a small piece of 3MM, add a drop of “anti-fade” mounting medium to the well and cover with a round coverslip 11mm diameter.
12 Safety issues:PFA (paraformaldehyde) is skin and airway irritant and could cause allergic reactions! Wash exposed skin areas with water and soap.May Grunewald staining solution is flammable and skin irritant. Rinse with ethanol followed by water in large amounts!Giemsa is skin irritant. Rinse with ethanol followed by water in large amounts!PI (propidium iodide) binds to DNA and should be handle with care. Rinse with ethanol followed by water in large amounts!All drugs are cytotoxic drugs. Rinse with ethanol followed by washing of exposed areas with water and soap.
13 Group wise presentation: Fully planned experimentsReagents and supplies and amount needed as well as a time scheduleDrugs: 200x final conc.Jurkat cells: Amount of cells and RPMI % FCS.Reagents : MT-STAB, MT-FIX, PI-solution, trypan blue solution, resazurin 4.4 mM, saponin 10%, antibodies, buffers……………………………………………..Other suppliesTubes Epp-tubes, multiwell plates, slides, cover slips …………………………………………….Equipment:You do not need to include pipett tips , disposable pipets, May Grunewald and Giemsa staining solutions and mounting media.Carefully planned time schedule for Monday 10/3 Tuesday 11/3.Calculations how to obtain original cell conc when cells are diluted 200ul cells + 50 ul trypanblue.
14 Format for seeding cells into 96-well plate for resazurin Time Slots for group discussions:Monday 10/3group8: , 16, 178:30 12,13, 149:00 9, 10, 1112:00 6, 7, 812:30 3, 4, 513:00 1, 2CBS1S2S3S4S6S7S5C Solvent controlS1 Cells treated with drug #1S2 Cells treated with drug #2and so onB culture medium without cells