Lab 3 Cell Biology 2015, Kristina Ruuth Characterisation of novel anticancer drugs PART 2 Most currently used anticancer drugs work by targeting the cytoskeleton, cell cycle progression or the apoptotic machinery. Tumor resistance to anti-cancer drugs Ref Per Holmfeldt/Mikael Sellin, Umeå University

Lab3: Each group will test 7 substanses from a chemical library plus 1 solvent control on Jurkat cells Cell 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

Cell viabilty: Trypan blue exclusion: Counting of trypan blue stained cells in a burker chamber. Count all samples in triplicate at 2 time points , 24 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/ml Diluted 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.

Metabolic activity, Proliferation assay. Cell Viability Metabolic activity, Proliferation assay. Many assays: MTT, WST, 3H-thymidine, Brdu incorporation, Resazurin. 590nm 530nm Resazurin (Alamar Blue): Medium + resazurin Untreated cells + Analyse all samples, in triplicate wells. Seed 150 uL/well, according to template, 96-well plate, 0.5x106 cells/mL Incubate 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 on Tuesday 10/3: 12:00-13:00, 14:00-15:00, 16:00-17:00

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 100-150 cells.

Methods to distinguish out stages in the cell cycle: G0 from G1. Ki-67 is not expressed in G0 cells G0/G1 S G2/M R Has cells passed the G1 restriction point. Western blot of cell lysates with Rb-antibodies Rb pRb actin

How to resolve different stages within G2/M G0/G1 S G2/M

FACS analysis of micro-tubules Jurkat cells Ref Dipak M et al Nature Comm 2013 μ-tubules Control Drug Intact cells Perforation of plasma membrane in presence of saponin and taxol; Unpolymerised tubulins will diffuse out of the cell Ref: Per Holmfeldt, Umeå university After fixation incubate cells with anti-tubulin antibodies followed by incubation with FITC-labelled secondary antibodies.

Staining of microtubules for FACS (copied from “-MG lab methods 2011-“) Spin down 500.000 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 at 37 °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-5-1-2 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-STAB PEM buffer, pH 6.9 0.1 % 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) BLOCK 90 % PBSA / 10 % FCS / 0,1 % Saponin WASH PBSA / 0,1 % Saponin PI SOL. PBSA 10µg/ml Propidium Iodide 0,1% Triton X-100 (add 1/100 of a 10 % stock) 10µg/ml RNase

micro-tubules är very britle Extraction of “free” tubulin Add 400 μL MT-STAB 37° inc 37° 4 min. Fixation of cells Add 400 μL MT-FIX 37° inc 37° 12 min Loosen pellet by gently shaking on shakerboard 500 μL cells Aspirate medium Centrifuge 1100 rpm 3 min Centrifuge 1100 rpm 2min Add 150 μL Mouse anti-α–tubulin Antibody and inc 60min 37° in shaking waterbath. Blocking of unspecific protein binding sites. Add 1ml BLOCK and inc 10min 37° Add 1 mL WASH Centrifuge 1100 rpm 5 min Aspirate sup and shake gently Aspirate sup and shake gently Centrifuge 1100 rpm 5 min Add 100 μL Secondary ab: FITC Rabbit α mouse IgG, inc 30min 37° in shaking waterbath Add 0.5ml PI-soution. Store cells in fridge until FACS analysis Aspirate sup and shake gently Add 1 mL WASH Centrifuge 1100 rpm 5 min Aspirate sup and shake gently

Structural effects on microtubules After FACS analysis of cells stained for u-tubules Adjust 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 wells an alternative to cytospinning Adjust 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.

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.

Group wise presentation: Fully planned experiments Reagents and supplies and amount needed as well as a time schedule Drugs: 200x final conc. Jurkat cells: Amount of cells and RPMI1640 10% FCS. Reagents : MT-STAB, MT-FIX, PI-solution, trypan blue solution, resazurin 4.4 mM, saponin 10%, antibodies, buffers…………………………………………….. Other supplies Tubes 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.

Format for seeding cells into 96-well plate for resazurin Time Slots for group discussions: Monday 10/3 group 8:00 15, 16, 17 8:30 12,13, 14 9:00 9, 10, 11 12:00 6, 7, 8 12:30 3, 4, 5 13:00 1, 2 C B S1 S2 S3 S4 S6 S7 S5 C Solvent control S1 Cells treated with drug #1 S2 Cells treated with drug #2 and so on B culture medium without cells