Sean Lauber:Flow Cytometry

Tissue preparation for FACS

1) After lung samples were BAL'ed, they were placed into 500 ul of PBS on ice. BAL stored at -80*C

2) Lungs were cut into smaller pieces with scissors

3) Lungs were transferred to 10 ml of HANKS buffer containing collagenase A (15 units/ml, CAT 17100-017, Life tech)

4) Incubate samples for 1 hour at 37*C

5) Pour samples into 40 um filter (blue) sitting on a 50 ml falcon tube and allow to drain

6) Begin grinding the lung tissue through the filter using the flat end of a 1 ml syringe plunger until it looks like paste

7) Add more HANKS to fill the filter and let drain

8) Remove the filter, cap the tube and spin at 1500 RPM for 10 min

9) Discard the supernatant and add 1 ml (100 ul for BAL) of ACK red blood cell lysis buffer

 For 500 ml ACK:
   4.145 g	ammonium chloride
   0.5 g	potassium bicarbonate
   18.6 mg	Disodium EDTA
   500 ml	water
 pH to 7.2-7.4. Filter sterilize and store in 100 ml aliquots at RT	

10) Incubate for 3 minutes at RT shaking

11) Stop the reaction by adding 9 ml (900 ul for BAL) of PBS

12) Spin at 1500 RPM for 10 min then discard supernatant (10 K for 2 min for BAL)

13) Add 500 ul (100 ul for BAL) of FACS Buffer (PBS/0.1% BSA) to each sample

14) Use a p1000 to pipette the sample until well mixed

15) Strain sample through another 40 um (blue) filter using the pipette

16) Transfer sample into eppendorf, count cells and use for FACS

Blocking and Staining

1) After counting the cells, take volumes required for 1x10^6 cells and put this into a round-bottom 96-well plate

2) Spin the plate down and discard the supernatant (gently blot the plate onto paper towels)

3) Add 50 ul of of FACS buffer containing 1 ug/50 ul of Fc block (BD, CAT 553141) and incubate at 4*C for 15 min

 For 50 ul block: 1 ug/500 ug/ml (stock) = 2 ul

4) To stain for surface antigens:

 a) Wash cells by first spinning them down (1500 RPM for 3 min), then   resuspending in 150 ul of FACS buffer, then spin down again and discard supernatant
 b) Add 50 ul of your diluted surface stain (in FACS buffer) and incubate at 4*C for 40 min in the dark
 c) Wash the cells

5) If a secondary Ab (ie. Streptavidin) needs to be added here, do it now (30 min at 4*C in the dark), then wash

6) If you need to stain for an intracellular antigen (that is not secreted):

 a) Resuspend washed cells in 100 ul of Fix/Perm solution (BD, CAT 554715) in the dark for 30 min at 4*C (fixation)
 b) Wash cells in 150 ul of 1X Perm/Wash buffer
 c) Add diluted stain (in Perm/Wash)
 d) Wash the cells

7) If you need to stain for intracellular cytokines add Ab diluted in PermWash (30 min at 4*C) then wash

8) Prepare the compensation beads (BD, CAT 552845 = anti-mouse beads)

 a) Combine 1 drop of the green cap and white cap into a tube
 b) Add 1 ul of stain and vortex
 c) To prepare a negative control tube, add a drop from the white cap bottle to a new tube
 d) Wait about 5 minutes before loading onto machine

9) Resuspend the washed cells in 200 ul FACS buffer then pass through a 0.4 um filter (blue cap)

10) Keep tubes on ice and covered in aluminum until ready to load into the machine

Using the BDCanto

Machine needs to be turned on:

1. Turn on machine using green button on left of machine

2. Check all the levels of the fluids below the machine

 Check by weight or eye
 If the sheath buffer or other buffer needs filling, fill a graduated cylinder with sheath and pour this into container (check under sink)
 If need to fill with distilled water, disconnect the unit (push-click one of them, simply pull the other one) and fill with distilled water in sink. Or use a squirt bottle.
 If need to empty waste (full or near 10 L), disconnect and dump in sink (pull cap off – don’t unscrew) (let water run 3 min) then fill with 1 L bleach
 To reconnect the wires, line up the arrow and push in for one of them. For the other just push-click it.

3. Login to computer

4. Let machine warm up for 30 minutes

5. Run Fluidics startup (cytometer/fluidics startup)

6. Degas the machine to clear bubbles (cytometer/cleaning mode/degas)

7. Load a water sample and run through machine for two minutes (acquire data, collect at HIGH)

8. Proceed to compensation

Machine already turned on:

1. Ensure green button on left of machine is on

2. Check levels as indicated above

3. Login to computer

4. Prime machine (cytometer/cleaning mode/prime)

5. Load a water sample and run through machine for 2 minutes (acquire data)

Compensation

1. Highlight the yellow folder

2. Create a new experiment

3. Highlight the experiment and create a new specimen

4. Press the acquisition control pointer on any tube and go to the Cytometer window. Go to the Parameters tab and delete any colors you don’t need (max=8). Select “W” for FSC and SSC to collect data for these parameters.

5. Select Experiment/Create Compensation Tubes

 New specimen with compensation tubes appear along with plots in the worksheet
 Create grids for each compensation plot

6. Select the acquisition pointer in front of the Unstained Tube

 Load the unstained sample into the machine and acquire data
 Adjust the Parameters/FSC and SSC voltage (in Cytometer window) in order to capture all events – RECORD YOUR VOLTAGES 
 Adjust the voltages for each stain to put the negative peak in the middle of the first quadrant, and coming down to end halfway into the 2nd quadrant
 Stop acquiring and remove the unstained sample (Remove Tube – during this time you must remove the tube quickly and snap back the receiving arm to trigger the wash)

7. Click next tube and the acquisition pointer will go to the next sample (first stained comp bead)

 Load your first stained comp bead sample, acquire data
 Using the plots in the worksheet area, make sure the positive signal is between 104-105. If it isn’t, adjust the voltage in Parameters to make it fall in this region. RECORD YOUR VOLTAGES.
 Stop acquiring and go to the next comp bead sample

8. If at any point you need to change one of the voltages of a previous compensation, you need to go back and change that voltage for those tubes before recording. When you start recording you MUST have all the voltages the same for each tube.

9. Now that you have all your voltages you need to record the events (5000 events) to calculate compensation

 First load the unstained comp bead sample, acquire for 5 seconds, then click record. Ensure (using worksheet plots) that everything looks okay
 Then record the next comp bead sample…After recording, the P2 gate should snap to the positive peak, if it doesn’t then try recording again. Remember it must be below 10^5

10. To calculate compensation go to Experiment/Instrument Setup/Calculate Compensation

11. Go to the Cytometer window and check that the spectral overlap for compensation doesn’t exceed 100

12. Link and Save

Loading samples

1. Now that you‘ve setup compensation, select your specimen outside of the compensation specimen and click the acquisition pointer on the tube in that specimen. Go to the acquisition dashboard and adjust the parameters you’d like for collecting events

 Usually you want to collect 10,000 events (display 1000). If you have few cells you might want to collect 50,000

2. Now begin clicking “Next Tube” until you have added many tubes as you’ll need

3. Create the plots

 In the worksheet, click “global worksheet” and begin drawing plots. Make as many colors as you have plus one.
   The first plot should be SSC-A (y-axis vs FSC-A (x-axis)
   The next plot will be SSC-A vs color 1, then color 2, etc.

4. Next load your sample into the machine and acquire for 5 seconds (ensure that events are being detected) before clicking Record. Try to collect about 2000 events/second, adjust the flow rate (low, medium, high) to achieve this. Once all the events are collected, Remove Tube (and then remove tube and snap arm back), then click Next Tube to go to the next sample…

Exporting Data

1. Once you‘re done, right click on the experiment folder you’ve recorded your data in and Export FCS. Make sure it’s in the FCS 3.0 format and go for it. Save it to your folder, and then use a USB to collect the data and delete it.