ENCENDIDO Y LAVADOS DEL CITÓMETRO

ENCENDIDO Y LAVADOS DEL CITÓMETRO ANTES DE INICIAR EL PARATO SI EL ORDENADOR ESTÁ ENCENDIDO Y EL CITÓMETRO APAGADO: 1. APAGAR EL ORDENADOR 2. ENCENDER EL CITÓMETRO 3. ENCENDER EL ORDENADOR AL INICIAR EL APARATO 1. Encender el transformador 2. Verificar el nivel de fluidos del bidón de FACSFlow y del Waste. 3. Encender la bomba de fluidos (solo en el caso de contar con carro de fluidos). 4. Encender el citómetro. 5. Colocar la palanca de VENT VALV en posición de presurizar. 6. Pulsar el botón de PRIME dos veces para quitar burbujas (sin introducir la aguja en el tubo) 7. Encender el ordenador y abrir el CellQuestPro. Abrir ventana de STATUS y esperar a que el citómetro pase de Not Ready a Standby. 8. Abrir plantilla 8.1 Cell QuestPro Adquirir Connect to Citometer FileOpen Document Buscar laboratorio y ubicación. 9. Cargar Setting Citometer Instruments Settings Buscar laboratorio y ubicación SET DONE Ya se puede empezar con la adquisición. AL FINALIZAR LA ADQUISICIÓN 10. Poner un tubo de FACSSafe y pasar durante 5 min. en HI con el brazo móvil CERRADO. 11. Poner Un tubo de FACSRinse y pasar durante 5 min. en HI con el brazo móvil ABIERTO. 12. Poner Un tubo de FACSRinse y pasar durante 5 min. en HI con el brazo móvil CERRADO. 13. Poner un tubo de H2O miliq y pasar durante 5 min. en HI con el brazo móvil CERRADO. 14. Una vez finalizados los lavados hay que dejar SIEMPRE el citómetro con un tubo con H2O miliq y No con FACSFlow para evitar precipitaciones de sales en la aguja. 15. Dejar el citómetro en Standby y DESPRESIRIZADO. 16. Si se han adquirido células con Ioduro de Propidio (PI) antes de comenzar los lavados del aparato es OBLIGATORIO adquirir de 3 a 5x10 6 células sin marcar, con objeto de retirar el PI que se ha quedado contaminando los conductos del aparato. Estas células pueden proceder de células en cultivo, células de ratón o cualquier otrto material disponible. Se recomienda fervientemente el uso de 7AAD en lugar de PI. SI HAY OTRO USUARIOI DETRÁS DE TI 17. Para evitar desgaste de los Láseres, mantener el aparato encendido siempre que haya una persona apuntada detrás de tí si el tiempo entre el fin de tu turno y el siguiente turno es < a 60 min.

SI ES EL ÚLTIMO USUARIO 18. Asegúrese de dejar el citómetro bien lavado y con la aguja en H2O miliq 19. Dejar el citómetro en Standby y DESPRESURIZADO. 20. Apagar el citómetro de flujo 21. Apagar la bomba de fluidos. 22. Apagar el transformador. 23. Apagar el ordenador conectado al cittómetro de flujo.

CYTOMETER, COMPUTER WORKSTATION, AND DATA MANAGEMENT Cytometer Anatomy FACSCalibur Optics Laser and Photomultiplier tubes (PMT s) 488 nm (FL1=515-545nm, FL2=564-606nm, FL3>670nm) 635nm (FL4=653-667nm) FL1 530/30 BP FL2 585/42 BP FL3 670 LP FL4 661/16 BP SSC 488/10 BP FSC diode 488/10 PMT Designation "FL1" "FL2" "FL3" "FL4" Filter Specs 530/30 BP 585/42 BP 670 LP 661/16 BP Fluorochrome s Fluoriscein (FITC) Phycoerythrin (PE) PerCP APC GFP Propidium Iodide (PI) PE-Cy5 Alexa 647 Alexa 488 7-AAD Cy5 CFSE PerCP- Cy5.5 ToPro-3 Electronics Boards Fluidics Fluidics Drawer Sheath reservoir (left) Waste reservoir (right) Vent toggle switch Sheath filter Tubing and wiring

FACSFlow supply system Waste cubitainer (left) Seath cubitainer (right) Start button Restart button Alarm Sample Injection Port (SIP) Sample injection tube/droplet containment system (DCS) Tube support arm Fluidics control panel LO (12 l/sec), MED (35 l/sec), HI (60 l/sec) sample flow rate PRIME, STANDBY, RUN

Software applications CELLQuest Pro Multipurpose data acquisition and analysis software. CELLQuest (old) Multipurpose data acquisition and analysis software FACSComp Quality control for instrument Used for setup with 2 nd laser FlowJo Multipurpose analysis application ModFit LT DNA analysis software. Data management 1. CELLQuest Pro Files Organization of files: Suggested file structure Data files folder Experiment document/template folder Instrument settings folder

START-UP AND CLEAN-UP PROCEDURES NOTE: If computer is ON and FACSCalibur is OFF, then shut down computer, then perform start-up as above. This enables the computer to recognize that the cytometer is connected. 1. Encender el transformador. 2. CHECK sheath and waste cubitainers. Full Waste: Exchange Cubitainer with an empty one; add 1l of Bleach to the full Cubitainer. The waste can be discarded into the sink at the next day. Empty Sheath Cubitainer: Exchange with a new one. 3. Switch the FACSFlow Supply system ON, press the Restart button: The system ON indicator will light. Press and hold the Prime button of support system until no air is left in the Sheath supply line. 4. Turn FACSCalibur ON (wait 30 seconds). 5. PRESSURIZE the sheath and sheath filter lines. Flip toggle toward you. 6. Set sample flow rate to HI. REMOVE dh 2 0 tube. Press PRIME (wait for instrument to go to standby), REPEAT. 7. When STANDBY button turns orange, then install dh 2 0 tube and place support arm under the tube. 8. Turn computer ON. Cancel or connect to server. 9. ACQUIRE samples after 5 minutes warm-up. When you are finished Cleaning Calibur 1. 2. Place FACS Clean solution in sample tube. Put support ARM UNDER the tube and RUN on HI for 5 MINUTES. 3. Place FACSRinse solution in sample tube with support ARM TO THE SIDE and RUN on HI for 5 MINUTE. 4. Put support ARM UNDER tube and RUN on HI for 5 MINUTES. 5. Place ~3ml dh 2 0 in another sample tube. Put support ARM UNDER tube and RUN on HI for 5 MINUTES. 6. Leave <1ml dh 2 0 on instrument. 7. Switch cytometer to STANDBY mode.

If someone else wants to use the machine directly after you: Note: If you are not sure if someone else is really going to use the machine within ca. 90min after you have finished, proceed with 10 instead 8. Leave instrument in Standby mode. 9. Close Cell Quest software. If you are the last user: 10. VENT the sheath tank (depressurise!!!) 11. Shut Down instrument 12. Shut Down FACS Supply system 13. Shut Down transformador 14. Shut Down computer If necessary: 15. Exchange SHEATH Cubitainer Container if empty. 16. EMPTY WASTE cubitainer if filled: Put undiluted Bleach in full cubitainer. Fresh Waste has to can go in the sink after incubation with bleach(overnight).

ACQUISTION AND ANALYSIS OF DATA USING CELLQUEST Acquisition of samples for phenotypic analysis A. CREATE or modify an acquisition experiment document. 1. Launch CELLQuest From the Dock (a new untitled experiment document is now open) 2. Create an acquisition dot plot with FSC and SSC as the x and y parameters and no gate. How?: Select the dot plot tool from the tool bar, click and drag on the document to create a plot of any size.

To modify a plot, select the plot and go to the plots menu and choose format plot. Then modify the plot. 3. Create an acquisition histogram plot for a single fluorescent parameter (FL1, 2, or 3) AND/OR Create an acquisition dot plot for dual parameter acquisition (e.g. FL1vs. FL2). How?: Go to the plots menu and choose your plot type and select the appropriate parameters. The plot you create will be the same size as the first FSC/SSC plot you created. 4. Modify each plot so that you have FSC vs SSC and a FL1 vs FL2.

B. SETUP for acquisition: optimize instrument settings and define regions. 1. Choose Detectors/Amps, Threshold, and Compensation from the Cytometer menu and place them on the screen at a convenient location. Detectors/Amps: This window indicates the voltage and amp values set for FSC, SSC, and each FL channel, which is determined for each type of experiment. Threshold: This value determines the lowest FSC limit for data acquisition. It is used to help eliminate dead cells/debris from your acquired events; any event falling under this value will not be collected by CellQuest. Compensation: This window sets values to correct for signal overlap between fluorescence channels. These values must be set properly for accurate results in 2 or 3 color experiments. (In general the instrument settings menus (detectors/amps, threshold, and compensation are dealt with in order, i.e. first detectors /amps are adjusted, then threshold, then compensation). Remember compensation adjustments are dependent upon the voltages applied to the different PMT s.) 2. Choose Instrument Settings from the Cytometry menu.

3. Click Open, go to Desktop/ Lab Temp Instrument Setting folder/calib file and choose the Calib file. 4. Click Set and then Done on the Instrument Settings window. Note: Instrument settings can also be retrieved from data files: Simply select the data file with the appropriate settings (while in instrument settings ), then click, Set and done. 5. Place Tube 1, subclass control (i.e. negative control), on the flow cytometer, select RUN on the cytometer and click Acquire. Make sure that SETUP is marked 6. Adjust the FSC amplifier, SSC detector in the detectors and amps window, so that the bead population is located in the center of the plot. The FSC and SSC amplifiers in general should be linear (exceptions for example, are red blood cells or platelets). Think of the forward scatter adjustment as focusing on a microscope. You have a course adjustment (voltage: E00, multiplies FSC signal by 1; E01, multiplies FSC signal by 10; E02, multiplies FSC signal by 100; E03, multiplies FSC signal by 1000; E-1, multiplies FSC signal by 0.1) and fine adjustment (amplifier 1.00-9.99). For side scatter and the fluorescent parameters adjust the voltage. The idea is to clearly identify your population on the screen. The more you know about the relative size and complexity of your cells, the better. 7. Adjust the FSC Threshold to eliminate extraneous events, noise, or debris. Thresholding is electronic gating. Events below a threshold value are electronically excluded from acquisition. FSC is the usual threshold parameter. 8. Define a region by selecting one of the region tools (rectangle, ellipse, or polygon) on the tool palette. 9. Draw a region around the events of interest. Format relevant plots with the defined region. Select a plot, go to the plots menu and choose format. Turn the gate on in the plot source menu. Adjust FL1 and FL2 detector voltages. 10. Remove Tube 1 from the cytometer and place Tube 2 (FITC positive control) on. 11. Click Acquire.

12. Place the population in the first log decade by adjusting the appropriate fluorescent detector voltages. You may want to place a quadrant marker for reference purposes. 13. Click Pause and Abort. 14. Repeat steps 10 thru 13 with Tube 3 (PE control). Fluorescence Compensation the FITC. This is necessary for appropriate multi-color analysis (NOT for single color analysis like EGFP alone). For a given setup of cells this is performed once initially, and does not need to be performed again unless antibody conjugates and/or combinations are changed, or, if laser, PMT or optical filter components or settings are modified. PE (tube 3) FITC (tube 2) 15. Place Tube 2 (FITC positive control) on. 16. Click Acquire. 17. Place the population in the first log decade by adjusting the appropriate fluorescent detector voltages. You may want to place a quadrant marker for reference purposes.

18. Click Pause and Abort. 19. Once the FITC is compensated, repeat steps 15 thru 18 with Tube 3 (PE control). 20. After samples are optimized, click Pause, then Abort. 21. Place a tube of DI water on the SIP and place the instrument in Standby. 22. Close Detectors/Amps, Threshold, and Compensation windows. C. ACQUIRE samples 1. Define where to save the data by selecting Directory Change button. 2. Navigate to the Desktop and create a new folder. Once created, select this folder. 3. Change the file name by first selecting on the File Button. 4. In the File window, change the prefix pull down menu to Sample ID. 5. Close the File window and type in a sample name in the Sample ID text box. 6. Type in labels in the Parameter Settings, so that you have labels on each of your axes. Label your parameters Fill in the appropriate parameter label for each sample. Example: P1=FSC P2=SSC P3=FITC P4=PE P5=FL3 P6=FLA P7=FLW or FL4 Time 7. Click on Acquisition and Storage Define Acquisition and Storage (WHAT data to acquire and save). Go to the Global Settings folder and open. Work from the top down in the menu and decide what data and parameters you want to acquire: For example: How many events to save Saving Time as a parameter: Define to save Events or Time

(Set time to for example 500 seconds ( The aquired data will be saved after reaching the selected number of cells or after 500 seconds). 8. Choose Counters from the Acquire menu and place on the screen. Expand the Counters window to full size. Counters: This window monitors event count (number of cells acquired) as well as the rate at which events are acquired. Ideally, counts should fall somewhere between 200 1000 events/second. 9. Place the Tube 4 on the cytometer in the RUN mode. 10. Uncheck the Setup box in the Acquisition Control bar of the browser. 11. Click Acquire (The setup box should be unchecked). The sample will be acquired. After the beep, the data file is saved and the file number will increment. Remove the tube and continue with subsequent tubes. Be sure to label parameters or change sample description as needed. Note: The cytometer will continue to empty your tube until you unload it. D. When you are finished 1. Save your acquisition experiment document by selecting Save Document in the File menu. 2. Navigate to your folder on the Desktop and save it there. 3. Save your instrument settings, by selecting Instrument Settings in the Cytometer menu. 4. Select save and name your file, click done. 5. Complete the clean up procedure.

2. FOUR-COLOR FLOW AND USING THE RED DIODE (635nm) LASER Overview 3. The standard configuration for a FACSCalibur is a fixed 488nm argon laser. A second 635nm red-diode laser has been added and can be used in conjunction with the first laser for four-color acquisition of data. 4. You must optimize the instrument with biological samples for accurate acquisition and interpretation of data. Controls for compensation are critical due to the number of dyes being used. Instrument setup 5. Cytometer should be on and primed. 6. Perform Time-delay Calibration. 7. Prepare APC beads 8. Purchase APC beads from Becton Dickinson, cat#340487. 9. Add one drop of beads to 1ml filtered sheath fluid. 10. Open time-delay template from the apple menu and follow the directions in the template. 11. Optimize instrument settings with biological samples.

12. TROUBLESHOOTING 13. Make sure sheath tank is pressurized. 14. Purge the sheath filter and prime the fluidics. 15. Run bleach and water. 16. Do not use contour plots in acquisition. 17. See Chapter 7 in FACSCalibur System User Guide. 18. See CELLQuest Pro User s Guide.