Live Cell imaging of mitochondria on micropatterns
Contents
1. Created 2012 11 14 Live Cell imaging of mitochondria on micropatterns MO EXT 18 Material and reagents Supplier Comments quantities for one experiment Hela cells ATCC CCL 2 MEM GlutaMax Invitrogen 41090 supplemented with 10 FBS and 0 5 penicillin streptomycin FB 0 05 Trypsine EDTA 1X 25300 054 See PRO EXT 10 CYTOOchamber User Manual 20 nM 15 min treatment 25 mM 0 2 um Vented Blue Plug Seal Cap Recommended protocol Warm up 1 bottle of media and trypsin Rinse the flask with 10 mL of PBS Trypsinize 70 80 confluent HeLa cells by adding 2 ml of Trypsine in T75 flasks Incubate 5 min at 37 C Check under microscope for efficient dissociation Add 8 ml of 10 FBS supplemented media to inactivate the trypsin To ensure the dissociation of last cell aggregates thoroughly pipet up and down the cell solution Spin 4 min at 300g Remove the supernatant and add 10 ml of fresh media thoroughly pipet up and down the cell solution Count cells and prepare a cell solution at 40 000 cells ml in growth media Dispense 500 ul 20 000 cells per CYTOOchamber containing the appropriate CYTOOchips See appendix 1 for CYTOOchamber use Incubate for 2h at 37 C then add 2 5 ml of culture media was added Note This step is required for timelapse experiments starting immediately after seeding It allows HeLa cells to sediment to attach to the micropatterns and to spread entirely which is necessary t2. ght Representative pictures of the mitochondrial network on different type of large microaptterns as well as standard culture conditions are presented at the lower side B Class of mitochondrial networks Q Small M ai Large Small M ER Large sakt M edi n Large Small M edium Large Stri pS HPODEENENE Version 0 Page 2 sur 4 Live Cell imaging of mitochondria on micropatterns ca MO EXT 18 Troubleshooting Problems Solution proposed Mitochondrial network is abnormal in lf you use Mitotracker Green FM it can be toxic for high more than 10 of the cells nicely concentrations and or long incubations Do not exceed spread on micropatterns concentration of 20 nM during 15 minutes incubation See appendix 3 for more details Addition of Hepes 25mM pH 7 4 for long time videomicroscopy is often required to buffer pH variation Hepes does not affect mitochondrial integrity on micropattern Ifyou are not using Mitotracker Green FM check for potential toxicity due to the marker concentration incubation time etc Wash carefully the CYTOOchamber before use as residual ethanol or precipitated salts might have a toxic effect Please refer to Appendix1 Mitochondrial network seems to be The micropattern might be too small for your cell line condensed Appendix 1 CYTOOchamber use See PRO EXT 10 CYTOOchamber User Manual Appendix 2 Preparation of Mitotracker Green FM so
3. lution One vial 50 ug of Mitotracker Green FM is dissolved in 74 4 ul of DMSO to make a 1 mM stock solution Then it is diluted to 1 in PBS and 6 ul 20 nM of the last solution is provided per CYTOOchamber containing 3 ml of culture media Incubate 15 minutes before removing the media and add 3 ml of fresh one Appendix 3 Mitotracker effects on mitochondrial network shape In order to prevent some discrepancies due to the use of mitotracker on cells cultured on micropatterns we realized a dose response of Mitotracker Green FM concentration and time of incubation on 20 30 single cells using the Starter s CYTOOchips Mitochondria networks integrity was classified into two classes intact or fragmented As presented in figure 2 the use of Mitotracker Green FM treatment on live HeLa cells seeded on micropatterns induces few mitochondria network fragmentation It indicates that CYTOO micropatterns and CYTOO chambers are not toxic for cells Moreover the use of 20 nM Mitotracker Green FM treatment during 15 min do not induces stress to the cells Increased Mitotracker concentration and or time exposure highly favor a global toxicity to the cells Version 0 Page 3 sur 4 Live Cell imaging of mitochondria on micropatterns ca MO EXT 18 Representative picture of Crossbow 1600 um A nary Mitochondria network shape after 20 nM 15 min Mitotracker treatment mitotracker treatment 100 90 80 70 60 k on ee Representative
4. o set up your experiment Incubate for 23h45 at 37 C Add 6 ul of Mitotracker Green FM solution to the cells for 15 minutes 20nM final See appendix 2 for preparation Replace the media containing the Mitotracker Green FM with 3 ml of fresh media Place the CYTOOchamber in a controlled environment imaging system and acquire living cells humidified atmosphere 7 CO2 37 C A protocol to test mitochondria network shape on CYTOO micropatterns in fixed conditions is currently in progress Version 0 Page 1 sur 4 Created 2012 11 14 Live Cell imaging of mitochondria on micropatterns MO EXT 18 Typical Results Following the protocol described above 20 40 single cells were analyzed for the 12 different types of micropatterns present on Starter s CYTOOchips Mitochondria networks integrity was classified into two classes intact or fragmented As presented in figure 1 we observed that the mitochondrial network of HeLa cells is 100 intact except for Small and Large Discs with less than 10 fragmentation A 00 90 80 70 60 50 40 30 20 10 0 Disc Crossbow Fibronectin Class1 Intact Network Class2 Fragemented Network Figure 1 Integrity of micropattern networks after 24Hrs culture in CYTOOchamber A The graphic represents the percentage of intact and fragmented networks according to the size and type of microaptterns Integrity was also evaluated in standard culture conditions Fibronectin ri
5. picture of Crossbow 1600 um sae 100 nM 15 min Mitotracker treatment 20 10 0 20 nM 15 min 100 nM 15 min 200 nM 45 min Number of cells Sa S Crossbow Figure 2 Micropattern network integrity using increasing amount of mitotracker concentration and time of incubation Cells were cultured 24 hours on 1600 um crossbow micropatterns A The histogram represents the percentage of intact and fragmented networks according to the concentrationand incubation time of mitotracker treatment B and C Representative pictures of 20 and 100 nM Mitotracker Green FM 15 minutes incubation treatment are presented on panels B and C respectively Mitotracker Green FM is in green and micropatterns are stained using Alexa 546 fluorochrome and are presented as red Version 0 Page 4 sur 4
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