ANALYTICAL CYTOMETRY - PRACTICE 2018/2019 16. – 18. 1. 2019, IBP Teachers: Mgr. Stanislav Drápela, Mgr. Radek Fedr, Mgr. Karel Souček PhD. Group A Arcidiacono, Orazio Angelo, učo 478852 [MBBI], 1 kr., Fagherazzi, Paolo, učo 490520 [FYZZ], 1 kr., Kocandová, Veronika, učo 395010 [SPBI], 1 kr., Krchová, Michaela, učo 423226 [SPBI], 1 kr., Kvokačková, Barbora, učo 487629 [FYZZ], 1 kr., Group B Nevědělová, Kateřina, učo 451592 [SPBI], 1 kr., Pícková, Markéta, učo 411211 [FYZZ], 1 kr., Vacek, Ondřej, učo 423268 [FYZZ], 1 kr., Váňa, Petr, učo 108420 [SPBI], 1 kr., Day 1 (16.1.) A) B) 9 - 14 hod Intro Hela 8 Fucci cells – analysis using flow cytometry (Verse) and CM MLN-4924 treatment 14- 18 hod Úvod Hela 8 Fucci cells – analysis using flow cytometry (Verse) and CM MLN-4924 treatment Day 2 (17.1.) A) B) 9 - 12 Harvest and fixation of cells for proliferation and cell cycle analysis. Analysis using flow cytometry. 12-15 Hela 8 Fucci – analysis on CM Harvest and fixation of cells for proliferation and cell cycle analysis. Analysis using flow cytometry. 14-18 Day (18.1.) A) B) 9 - 13.30 Harvest of cells, immunophenotyping, analysis using flow cytometry 13. 30 - 18 Harvest of cells, immunophenotyping, analysis using flow cytometry Protokol 1 Fucci 8 cells – harvest, measurement, analysis of cell cycle using intracellular fluorescent proteins (both flow and CM) Protokol 2 Simultaneous analysis of proliferation and cell cycle on DU145 cells after the treatment by inhibitor of neddylation. Protokol 3 Immunophenotyping – staining of surface molecules CD24/CD44 and viability on DU145 cells. Protokol 1 Model HeLa 8 Fucci cells – cell cycle analysis using fluorescent proteins Aims - to demostrate how to analyse cell cycle w/o any fixation and staining steps on living cells using flow cytometry and confocal microscopy - analysis will be done on FACSVerse as one representative sample - evaluation will be performed in FlowJo software Theory Buněčná linie HeLa 8 Fucci - HeLa cells – human permanent cell line derived from cervical carcinoma - one of the oldest and most common cell model used in cancer research - Fucci probe (fluorescent ubiquitination-based cell cycle indicator) – enables visualisation of cell cycle progression in living cells - cells in G1 phase emits red light, cells in S/G2/M green light - find more info in PDF attached in your materials in IS (Sakaue-Sawano et al., 2008; materials) 1) Flow cytometry analysis of cell cycle Material - HeLa 8 Fucci cell line - solution of PBS+EDTA – disturb cell-to-cell junctions - trypsin – pancreatic enzyme - non-nesteril media with serum – for trypsin inactivation - PBS – for washing steps Process: Cell harvest and sample preparation - soak up the media from the dish - add 3mL of PBS+EDTA – 1-2 minutes than remove - add 0,5 mL of Trypsin – let incubate in termostat (37^oC) until the cells release from dish surface (cca 1-2 mins) - add 2,5 mL of media with serum - wash the dish with 1 mL PBS, add to the suspension into tube - centrifuge 200g, 5 mins - soak up supernatant - resuspend pelet in v 1 mL PBS - centrifuge 200g, 5 mins - soak up supernatant - resuspend pelet in 300 ml PBS and measure Results Describe the proces of measurement and analysis of cell cycle in Hella 8 Fucci. Attach results (plots) acquired from FlowJo evalutation. 2) Confocal microscopy analysis Process: Day 1: Seeding of HeLa 8 Fucci cell for CM analysis. Day 2: Treatment MLN-4924 (stock 10 mM, working concentration 1 µM) TRAIL (100 ug/ml stock, 50 ng/ml working concentration) Mitomycin (stock 1 mg/ml, working concentration 1 µg/ml) Add notes and descriptions to all drugs used - MLN-4924 (see protocol 2), TRAIL a Mitomycin (drug type, mechanism of action). Count dilutions and volumes which will be used for the treatment. Day 2-3: Analysis of cell cycle using confocal microscopy Describe the analysis of cell cycle using CM. Describe the changes in cell cycle, observed after various treatments. Protokol 2 Analysis of cell cycle, proliferation and cell viability on DU145 cells after the treatment by inhibitor of neddylation Aims - to describe the effect of neddylation inhibitor (MLN-4924) on DU145 cells - to use FACSVerse cytometr for sample analysis Theory MLN-4924 * ATP competitive inhibitor * Phase I of clinical trials for lymphoma, myeloma, AML, ALL, melanoma and other non-hematological cancers * creates stable aduct between NEDD8 and MLN-4924 which leads to the arres of neddylation pathway (figure Soucy et al., 2010). * proces of neddylation is necessary for the ubiquitine ligase Skp2^SCF aktivity which play role in the regulation of various cell cycle processes * one of the most important binding substrates are proteins regulating cell cycle (p27^Kip1, p21^cip1) or replication (Cdt1). Structure of MLN-4924 (Soucy et al., 2009) Neddylation pathway arrest (Soucy et al., 2010) Analysis of proliferation and cell cycle Material - DU 145 cell line (untreated vs treatred) - PBS+EDTA trypsin - nonsterile media with serum - nonsterile FACS tubes - PBS + 1% BSA - Live Dead Fixable stain kit Red - Edu click-iT AF488 kit - PO-PRO-1 Process 1. Cell harvest and sample preparation - soak up the media from the dish - add 3mL of PBS+EDTA – 1-2 minutes than remove - add 0,5 mL of Trypsin – let incubate in termostat (37^oC) until the cells release from dish surface (cca 1-2 mins) - add 2,5 mL of media with serum - wash the dish with 1 mL PBS, add to the suspension into tube - centrifuge 200g, 5 mins - soak up supernatant - resuspend pelet in v 1 mL PBS - centrifuge 200g, 5 mins - soak up supernatant 2. Viability stain - dilute viability marker in PBS (1:1000) - add 100 µl/sample, incubate 15 mins, 4°C - add 1 ml PBS + 1% BSA, centrifuge (200g, 5 min), soak up supernatant 2. Fix - resuspend cells in 100 µl 4% PFA - incubate 15 mins, RT - add 1 ml PBS + 1% BSA, centrifuge (200g, 5 min), soak up supernatant 2. Permeabilitation - resuspend cells in 100 µl 0,15% Tritonu X-100 - incubate 15 min, RT - add 1 ml PBS + 1% BSA, centrifuge (200g, 5 min), soak up supernatant 2. Click-iT reaction - divide saples into two tubes (ISO and SP) - prepare click-iT reaction solution based on recipe bellow - add 125uL of PBS + 1% BSA into ISO tubes; and 125uL of click-iT reaction solution into SP tubes - incubate 30 mins, RT, dark - than add 1 ml PBS + 1% BSA, centrifuge (200g, 5 min), soak up supernatant 1 reaction PBS 109,5 μl CuSO4 2,5 μl Fluorescent dye azide 0,625 μl Reaction buffer additive (dilluted) 12,5 μl Total reaction volume 125 μl 2. Cell cycle staining - dilute PO-PRO-1 in PBS (1:10 000) - add 500 µl/vzorek - incubate 30 mins, RT, dark Results Describe the process of measurement and analysis of results acquired using flow cytometry. Attach result plots from FlowJo evaluation. Protokol 3 Analysis of DU145 cell phenotype Aim - to analyse DU145 cell phenotype using two surface molecules CD24 and CD44 (primary Ab) conjugated with fluorescent probes on living cells Theory § DU-145 model is epithelial cell line derived from prostate cancer brain metastasis * CD24 and CD44 are characteristic markers of cancer stem cells (CSC) in prostate cancer * CSC – cancer cell subpopulations responsible for progression of disease and metastasis * CSC traits – self-renewal, increased expression of antiapoptotic molecules, expression of molecules responsible for multidrug resistance (ABC transporters) etc. CD44 * surface molecule associated with proliferation, differantiation, migration and angiogenesis processes * associated with worse prognosis in various types of malignancies * ligands – osteopontin, fibronectin, collagen, hyaluronate * in prostate cancer considered as a marker of cancer but also normal stem cells CD24 § surface molecule § marker of nondiferentiated hematopoetic cells § play role in cell adhesion § acts as receptor for P-selectin § increased expression shown in breast, ovarium and prostate cancer Material - DU-145 cells - solution of PBS+EDTA - trypsin - nonsterile media with serum - nonsterime FACS tubes - PBS + 1% BSA - antibodies – table bellow Count: for 10 ml 1% BSA add ml 20 % BSA into ml PBS Antibodies: antibody fluorochrom provider, cat. number dilution CD24 CD44 viabilita IgG2a κ IgG2b Samples: - 2 samples: specific (SP) isotype control (ISO) Process: 1. Sample preparation - soak up the media from the dish - add 3mL of PBS+EDTA – 1-2 minutes than remove - add 0,5 mL of Trypsin – let incubate in termostat (37^oC) until the cells release from dish surface (cca 1-2 mins) - add 2,5 mL of media with serum - wash the dish with 1 mL PBS, add to the suspension into tube - centrifuge 200g, 5 mins - soak up supernatant - add 1 ml PBS+1% BSA - both sample divide into 2 tubes - centrifuge 200g, 5 mins - soak up supernatant 2. CD24 a CD44 staining - add 100uL of antibodies or isotype controls diluted in PBS+1% BSA Count: 1. tube ISO – into 50 ml PBS+1% BSA add ml IgG ml IgG 2. tube SP – into 50 uL of PBS+ 1% BSA add ml CD44 ml CD24 - pippete the sample up and down twice - incubate 20 mins in 4C - add 1 ml PBS + 1% BSA - centrifuge 200g; 5mins - soak up supernatant 3. viability staining - rozsuspend in 500 ml PBS - add Propidium iodide (1:200) - measure Results Describe the process of measurement and analysis of results acquired using flow cytometry. Attach result plots from FlowJo evaluation.