Como parte de la residencia Prototyp_ome hemos estado dos días trabajando en el Departamento de Biología de la Infección (Andreas Meyerhans y Jordi Argilaguet) del Parque de Resercha Biomédica de Barcelona .
Workshop dado por Valentina Casella (Biología de la infección) ||| Con la traducción científica, el acompañamiento y las risas de Nuria Conde (DiyBio Barcelona)
Tanto las imágenes explicativas como los protocolos nos fueron dados durante el workshop. Dejamos aquí solo una selección.
Acerca del HPV y la Terapia Fotodinámica
Dia 1: Working with cell cultures: Cells passage (splitting) and counting
1) Prepare a new flask and put PBS, Trypsin, Medium (RPMI/DMEM+ 2-10%FBS) in the bath at 37°C for 10 min 2) Check cells at the Microscope
(In case of adherent cells ex. MC57)
3) Remove old medium from the flask 4) Wash with PBS two times 5) Add 1.5 mL of Trypsin for 1-2 min and put cells in the incubator at 37°C 6) Then hit gently the flask on the side to make it easier for the cells to detatch 7) Add medium to the flask and recollect cells in a falcon 8) Centrifuge cells at 600G for 6 minutes at room temperature 9) Remove supernatant 10)Resuspend cells in a falcon with new medium ( ex. 5 mL) 11) Count cells (see next protocol) 12) Add to the cells the amount of medium required in order to have them at the concentration desired (for ex. 1x106/mL) 13) Prepare a new flask with fresh medium and add cells
(In case of cells in suspension ex. Jurkat)
3) Recollect medium from the flask in a falcon 4) Centrifuge cells at 600G for 6 minutes at room temperature 5) Remove supernatant 6) Resuspend cells in new medium ( ex. 5 mL) 7) Count cells 8) Add to the cells the amount of medium required in order to have them at the concentration desired (for ex. 1x106/mL) 9) Prepare a new flask with fresh medium and add cells
Cells can be counted using a counting chamber, also known as Hemocytometer, which is a slide with gridded chambers, covered with a glass slide.
1. Cells from cell culture are diluted and mixed with Trypan blue (mark viable cells):
- prepare a eppendorf with 90 μL medium+10 μL cells and mix (dilution 1:10)
- take 10 μL from this dilution and mix in a new eppendorf with 10 μL L of Trypan blue (dilution 1:2)
So now we have a total dilution factor of 20 (10x2)
2. Prepare the hemocytometer with the slide of glass placed over it and transfer 10 μL from the last mix, in the space in-between the chamber and the glass.
3. Looking at the sample under the microscope, cells are counted manually using the grid (count cells in the four grids)
(Cells counted in 1+2+3+4) X 104 X dilution factor X mL of cell culture / 4
Día 2: Experiment. Campthotecin treatment to induce apoptosis in a dose-dependent manner
Jurkat cells are maintained in the culture medium RPMI, supplemented with 10% fetal bovine serum (FBS), 1% Pen/Strep and Glutamax.
1. Prepare cells in fresh RPMI medium at a concentration of 1 x 106 cells/mL in desired tubes: (we prepare 7 tubes with 1 x 106 cells each)
- Check cells at the microscope
Collect the cells from the flask in a falcon and centrifuge at 300g for 6 min at room temperature Discard supernatant and add 10 mL of medium Count cells Add amount of medium necessary to have 1 x 106 cells/mL Add 1mL to each corresponding tube (1 x 106 cells)
A 10-mM stock solution of Camptothecin was prepared in dimethyl sulfoxide (DMSO) and diluted to final concentrations of 0.1, 1 ,10 and 50 μM into RPMI medium.
2. Add an appropriate amount of 10 mM Camptothecin to the cell suspension(1x10^6) to achieve a final concentration of 0, 0.1, 1.0 and 10 μM:
Centrifuge the tubes at 300g for 6 min at room temperature Discard supernatant Add 1mL of Camptothecin 50 μM to tube ‘50 μM’ and resuspend cells pipetting Add 1mL of Camptothecin 10 μM to tube ‘10 μM’ and resuspend cells pipetting Add 1mL of Camptothecin 1 μM to tube ‘1 μM’ and resuspend cells pipetting Add 1mL of Camptothecin 0.1 μM to tube ‘0.1 μM’ and resuspend cells pipetting Add 1mL of RPMI medium to tube ‘Untreated’ and resuspend cells pipetting
3. Incubate cells at 37°C, 5% CO2 for 4 hours.
Apoptotic cells staining with Annexin V and Propidium Iodide
4. Centrifuge the tubes at 300g for 6 min at room temperature and discard supernatant
5. Wash two times with PBS:
Resuspend cells with 1mL PBS Centrifuge the tubes at 300g for 6 min at room temperature Discard the supernatant Resuspend cells with 1mL PBS Centrifuge the tubes at 300g for 6 min at room temperature Discard the supernatant
6. Resuspend cells with 1mL of 1X Binding buffer
7. Transfer 300 μL of this solution to new FACS tubes
8. Add 15 μL of FITC Annexin V and 15 μL of Propidium Iodide (PI)
9. Vortex and incubate at room temperature for 15 minutes in the dark!
10. Add 400 μL of 1X Binding buffer to each tube
Analyse at FACS within 1 hour!
Células tratadas por las Quimeras en proceso de apoptosis (induciendo suicidio en masa)