FACSAria I Standard Operation Protocol Basic Operation

Transcription

1 FACSAria I Standard Operation Protocol Basic Operation 1. Checking Lasers Status a. Please check the ON / OFF of the lasers. Sufficient time (~30 minutes) need to be given to allow the laser(s) to warm up completely. b. Lasers NOT required for analysis and/or sorting need to be switched OFF. 2. BD FACSDiva Software Log In a. Log into FACSDiva software with your own login name and password. Please contact the administrator to establish a new user account. b. When connected, click Use CST Settings whenever you see the below pop-up message. BD FACSAria I Operation Manual 1

2 3. For Sorting: Stream Break-Off and Droplet Formation (100µm nozzle) a. Turn ON the Stream and allow time for it to stabilize. b. Adjust the Frequency (Freq) between 27 and 30 to identify the HIGHEST break-off, where the End-portion (bottom neck) of the breaking stream can still be observed. c. Adjust the Amplitude (Ampl) to introduce DROP 1 measured between 120 and 180; but repeat the search using a different Frequency if the finishing Amplitude value is greater than 20. d. Fine tune the Amplitude value to stabilize the GAP measured between 8 and 12. e. Ensure that the satellite droplet has completely merged with the trailing drop (before disappearing). f. Match the input number with the measured value in DROP 1. g. Allow the stream to stabilize for a minimum of 3 minutes before the activation of Sweet Spot. h. Ensure the stream has been stable for at least 3 minutes and that the Sweet Spot is turned ON. BD FACSAria I Operation Manual 2

3 Drops Too FEW Drops Too MANY Break-off Too SOON Solution: Increase Freq Solution: Decrease Freq Solution: Decrease Ampl Gap Too NARROW Gap Too WIDE Break-off Too LATE Solution: Increase Ampl Solution: Decrease Ampl Solution: Increase Ampl BD FACSAria I Operation Manual 3

4 4. For sorting: ACCUDROP Drop Delay Test (100µm nozzle) a. Turn on Voltage Test Sort move voltage sliders to appropriate positions appeared similar to below. DO NOT touch the deflection plates when the plate voltage is ON!!! (When the voltage warning light illuminates, indicating that the plates are charged at 12,000-volt). b. Adjust the Micrometer Dial to obtain evenly bright bead spots for the four side streams. Core Stream Bead Spots TOO BRIGHT Bead Spots UNEVENLY BRIGHT c. Use the existing well-prepared Accudrop bead solution in the fridge. You are allowed to prepare fresh stock only if Accudrop solution has been used up (1 drop Accudrop beads + 350µL FACSFlow solution). d. Go to Browser Window Experiment New Experiment Accudrop Drop Delay OK. BD FACSAria I Operation Manual 4

5 e. Click + of Specimen_001, and set the tube pointer onto Tube_001. f. Make sure the flow rate is 1.00 followed by loading sample into the Bulk Injection Chamber (picture on the right). g. Adjust the flow rate to achieve ~1200 events per second; if failed, increase the flow rate or prepare suspension at a higher concentration. h. Go to Browser Window Create a new Sort Layout. i. Select 2 Tube in Device Initial in Precision right click on the Left Position and select Add > P1. BD FACSAria I Operation Manual 5

6 j. Click Voltage and Optical Filter on the Side Stream Window Sort on the Sort Layout Window. k. Click Cancel on the Confirm Prompt. l. Adjust the voltage slider and make sure the left side stream is contained in the box. m. Adjust the Drop Delay value (either or ) to achieve ~100% intensity in the left side stream. BD FACSAria I Operation Manual 6

7 n. When proximate to 100% intensity, change to Fine Tune. o. Optimize the drop delay again (either or ) until the left side stream intensity is consistently > 95%. Note: Two percentage values must add up to 100 %. p. Turn off the Voltage and Optical Filter. q. Click Sort to stop sorting; select Cancel when asked if preferred to save the report. Unload the tube. BD FACSAria I Operation Manual 7

8 5. Sample Collecting Tube(s) Alignment (FOR SORTING) a. For sorting, lift up the lid and insert the mock tubes into appropriate collection device. Holders are available for 1.5mL microtubes, 12x75mm tubes, and 15mL Falcon tubes. b. To keep the collecting tubes cool throughout the sorting process, collection device should be connected with tubings directed from the water bath. c. Click Voltage Test Sort Waste Drawer Open the sort block door Adjust the slider of the required side stream and locate the position for target cells collection. d. Turn off Voltage Test Sort Waste Drawer Close sort block door when alignment has been completed remove the mock tubes and replace new collection tubes with fresh media. BD FACSAria I Operation Manual 8

9 6. Creating A New Experiment a. Browser Window New Folder New Experiment. b. Select Cytometer Settings Parameters Delete the unnecessary parameters on the Inspector Window. c. FSC (measures Cell Size) and SSC (measures Cell Granularity) are MUST for all kind of analysis; their Area, Height and Width but not Log boxes should be ticked. Unless assigned for cell cycle and/or DNA analysis, Log and Area boxes of fluorescence channels must be ticked. BD FACSAria I Operation Manual 9

10 d. Choose Experiment Experiment Layout and define labels for each parameter. Select the column of fluorescence channel and enter a label in the Quick Entry Label field. e. Select New Specimen expands the Specimen to show Tube 001. Highlight the tube with the Tube Pointer. f. Select Dot Plot or Histogram move the cursor onto the blank worksheet. BD FACSAria I Operation Manual 10

11 g. Right click on a plot Duplicate to create another plot of the same type with identical axis. h. Select each individual axis, and opt from a list the preferred parameter. BD FACSAria I Operation Manual 11

12 i. Below shows a template of plots used in routine analysis. Dot Plot Dot Plot Dot Plot Histogram Histogram Histogram Dot Plot Dot Plot Dot Plot BD FACSAria I Operation Manual 12

13 j. Right click on the plot Show Population Hierarchy Right click on the plot Create Statistics View right click on the statistics view Edit Statistics View k. Select Statistics tab tick mean of FSC-A and FSC-H tick the mean of the A rea of the parameters click OK BD FACSAria I Operation Manual 13

14 7. Procedures for sample acquisition a. Put your sample tube onto the loading dock. Ensure that the flow rate is 1.0. Press Load on Acquisition Dashboard. b. Identify the population of interest by adjusting the voltage of FSC and SSC on Parameters Press Restart to accelerate the changes BD FACSAria I Operation Manual 14

15 c. Cytometer Laser Adjust the FSC Area Scaling until the mean of FSC-A and FSC-H are APPROXIMATELY SAME. Return to Parameters tab and finely re-adjust voltage of FSC and SSC. d. Adjust voltage of each of the fluorescent channels; preferably the peak of population of interest is greater than ZERO but less than e. Press Unload on Acquisition Dashboard to unload the current sample tube. f. Repeat above steps with the positive control sample tubes. Adjust the voltage of corresponding channels if their signal peaks are outside the limit of the histograms. BD FACSAria I Operation Manual 15

16 8. Creating Gates a. Set the current tube pointer to the following tube and repeat step l, m, o and p with the Unstained / Negative Control tube. Autopolygon Snap-to Polygon Rectangle Quadrant Interval b. Gate the cells of interest according to the following sequence by using: Create a polygon gate (P1) to gate the main population on the FSC-A vs SSC-A dot plot Highlight P1 population in Population Hierarchy Create a polygon gate (P2) around the singlet event on the FSC-H vs FSC-W dot plot Highlight P3 population under P2 Create a polygon gate (P3) around the singlet event on the SSC-H vs SSC-W dot plot Highlight P2 population under P1 BD FACSAria I Operation Manual 16

17 c. To define fluorescence positive signal, interval gate (P4, P5, ) beyond negative peak of fluorescent channels in histogram plot of unstained samples can be created. For over multiple fluorescence channels, quadrant gate could be created to define single/double positive signals (Q1; Q2; Q3; Q4.). *P1 is the children of All Events and the parent of P2 population; P2 population is the children of P1 population and the parent of P3 population and the grandparent of P4, P5, & P6 populations. On the hierarchy table, you should highlight the P1 population when a gate for P2 population is drawn, the P2 population when a gate for P3 population is drawn, and the P3 population when gates for P4, P5, & P6 populations are drawn. BD FACSAria I Operation Manual 17

18 d. Acquisition Dashboard click Next Tube to create a slot for new sample tube / to assign to the following sample tube slot. e. Ensure the Acquisition Setup has been arranged as follows: f. Acquisition Dashboard click Record Data to record entire sample set. BD FACSAria I Operation Manual 18

19 9. Setting up a Sorting Experiment a. Ensure that the flow rate is 1.0 before loading the sample tube. b. Browser Window Create a new Sort Layout. c. Determine the most appropriate options from Device and Precision on the Sort Layout Window. (i) For 2-12x75mm tubes sorting, select 2 Tube device (ii) For 4 15mL Falcon tubes sorting, select 4 Tube device (iii) To achieve the highest yield sorting, choose Yield (iv) To achieve the highest purity sorting, choose Purity in 2 Tube sorting and 4-way Purity in 4 Tube sorting. (v) To achieve single cell sorting, choose Single Cell BD FACSAria I Operation Manual 19

20 d. Right click Far Left / Left / Right / Far Right Position Add Gate of Interest Sort e. Click OK on the Confirm Prompt. f. Acquire data for the entire sample set. In each run, record all data by clicking Record Data. g. Constantly check for the sufficient level of samples inside the loading chamber and plenty of room inside the collecting tubes for sorted samples. Unload the sample tube and rinse the tubing with FACSRinse solution and Milli-Q water for five minutes each, if the threshold rate fluctuates high and low, and/or the stream is unstable. BD FACSAria I Operation Manual 20

21 Notes During the process of Sorting 1. To help achieve an optimal result, users are advised to pay attention to the following items: Sample: 5 tubes of 1mL sample, rather than 1 tube of 5mL sample Threshold Rate: Stable and no fluctuation Sort Rate: Stable and no fluctuation Stream: Stable and no sudden movement Side Stream: Stable and no fanning Sample Tube: Sufficient sample level to prevent clogging Collecting Tube: Plenty of room for collection 2. If required to change the collecting tubes Pause retrieve the fully filled collection tubes and replace with new tubes OK on the Confirm Prompt Resume 3. Upon completion, click Sort OK Unload the sample tube. 10. Cleaning the System a. Add 2 ml of FACSClean Solution, FACSRinse Solution, and Milli-Q H 2 O into each 3 individual tubes. b. Load each tube into the loading chamber. c. Increase the flow rate to 11.0 and let each solution run for 5 minutes. d. Repeat the same with FACSRinse Solution for 10 min, if Propidium Iodide (PI) is used. BD FACSAria I Operation Manual 21

22 11. Export FCS Data / Experiment a. To save Experiment, right click on the Experiment Export Experiment Browse to choose the destination folder. b. Create a new folder and rename, then click Export OK. c. To save FCS files, right click Specimen Export FCS Files. BD FACSAria I Operation Manual 22

23 d. Select FCS 3.0 OK Browse to choose folder destination. e. Create a new folder and rename Choose Directory Save. f. Delete experiment after having FCS data / experiment exported. 12. Log Out a. To log out of FACSDiva software, go to File Log Out. BD FACSAria I Operation Manual 23