User:Aelindor/Preparation for flow cytometry

Overview

The following example protocol is meant to illustrate a typical preparation in a lab for use with flow cytometry, complete with example-specific information.

Bold text indicates the objectives of and the general idea behind each step.
Plain text indicates a sample, detailed procedure by which this is accomplished, as well as ancillary information (ie. hints).

This is meant to be a complete, fully workable, step-by-step illustration of the process, one that can actually be used in the lab by a scientist new to the procedure. The particulars may vary depending on your objectives.

Typical duration of complete procedure:

For 2 tissue samples: 5-6 hours
For 4 tissue samples: 6-7 hours
For 6 tissue samples: 7-8 hours

Materials and Instruments

Generic lab equipment
- Pipettes w/ tips (ie. 20 PP, 200 PP, 1000 PP)
- Test tubes (ie. 1.5 ml, 15 ml) with rack
- Ice bucket (or refrigerator) (Ice or dry ice)
Solutions/reagents
- Staining buffer
- RBC lysis buffer
- PBS
- Trypan Blue (or other cell viability dye)
- Anti-CD16/32 antibody
- Primary antibodies (ie. B220, CD3e, CD4, CD8, CD11c, CD28, IgM, IgD, Gr1)
Instruments
- Centrifuge
- Vacuum aspirator
- Flow cytometer (+sheath fluid/bacteriostat)
- Microscope
Hemocytometer
Frosted microscope slides
Petri dish

Preparation

Harvest selected mouse tissue.
- Until used, tissue should be kept on ice (kept cold). For example, wrap tissue in aluminum foil and put on dry ice.
- You may want to save 1/3 of tissue for freezing at -80 C and 1/3 for fixing in 10% formalin (for other lab purposes).
Tease tissue apart using frosted slides, then put in 15 ml tubes.
- In a petri dish, add 1-2 ml staining buffer + selected specimen.
- Using the frosted side of the microscope slides, rigorously rub the specimen. The "front side" of a frosted slide is the side that's actually frosted, and the side you want to use.
- The tissue will disintegrate into its component cells and the liquid will turn reddish, resembling blood. The remains of the tissue - a spongy matrix - might remain. Try to thoroughly separate out the clumped cells.
- You won't be able to fully separate all the cells; neither do you need to. The process should only take a few seconds of careful rubbing.
- Return this teased tissue back to the petri dish, mix, then pour the contents (the cell suspension) into a 15 ml tube. Then place on ice, and start to work on the next one.
Wait a few minutes for the cell suspension to settle.
- If you're teasing five or more tissue samples, the first ones will have settled by the time you're done teasing the tissue.
- You will know when it's settled - a pellet (a solid chunk) will appear at the bottom.
Create single cell suspension.
- What you want is the cell suspension (the supernatant), not the pellet. Use a pipette to transfer as much as you can of the cell suspension of each sample into a second set of 15 ml tubes, without disturbing the pellet.
- What you now have in the second set of tubes is the cell suspension, plus some serum which you don't want.
- Centrifuge the second set of tubes at 400 G for 5 min at 4 C.
- Now the supernatant is serum and the pellet is the single cell suspension. Discard the supernatant using a vacuum aspirator. It doesn't need to be perfect.
If working with spleen, lyse red blood cells.
- If not working with spleen, skip this section.
- Add 2 ml RBC lysis buffer to each of the second set of tubes and resuspend the pellet.
- You can resuspend by A) pipetting up and down or B) flicking at it with your fingers. Do NOT vortex it or the cells will break.
- Wait 4 minutes. During this time the lysis buffer is doing its work (lysing red blood cells).
- Stop the reaction by adding excess PBS (fill each tube with about 12 ml of PBS, so that it's nearly full). The reason this works is because the lysis buffer works by osmosis, and its concentration is diluted by adding PBS.
- If there are visible chunks of cells in the tube, wait for them to collect at the bottom of the tube, then extract supernatant and put it in a new set of tubes, then centrifuge them.
Determine cell number.
- Resuspend the cells by adding 1 ml staining buffer.
- In a third set of test tubes, add 190 μl staining buffer (or PBS) + 10 μl of cell suspension + 2 μl Trypan Blue. This results in a 1:20 dilution.
- You can instead use other dilutions to make counting easier or to achieve a better accuracy cell count, but you must then alter other numbers in this protocol.
- Mix well, then pipette 10 μl of solution into hemocytometer slot. The solution should suddenly go all over the metal plate.
- View under microscope to determine cell density. (The 1 mm² (5x5) square corresponds to 100 nl. Since this is already 1:20 diluted, count the number of cells in this square and multiply by 2x10⁵, not 10⁴, to get cells /ml. So if you count 200 cells, the true density is 2x2x10⁷ /ml.)
Equalize cell concentrations.
- Add staining buffer (or PBS) to bring cell concentration to 2x10⁷ /ml.
- For example, if you had counted 200 cells in the grid in the previous step, this is what you want so do nothing.
- For example, if you had counted 100 cells in the grid in the previous step, the density is 10⁷ /ml, so you would then centrifuge, discard supernatant, then add half as much staining buffer (or PBS) as you had prior.
- For example, if you had counted 400 cells in the grid in the previous step, the density is 40⁷ /ml, so you would then double the amount of staining buffer.
- If you had counted different cell numbers, your tubes would have differing volumes by the end of this step, but they should appear to have the same color (since they are now of the same cell concentrations).
Block with anti-CD16/32.
- Here X = number of samples (number of tissues you're running your experiment on) and Y = number of antibody sets (reactions per sample) (number of sets of antibodies being run of each of your tissue samples).
- For each sample in the current set of tubes, transfer Y*50 μl of cell suspension into a new tube (4th set).
- Add Y*0.5 μl of anti-CD16/32 (the blocking antibody) to each tube.
- Incubate for 5-10 min on ice. Now the antibody is attaching to the cells.
- Wash the 4th test tube set. (Add 1 ml staining buffer, resuspend, centrifuge (400 G, 5 min, 4 ºC), discard supernatant.)
- Remember, resuspend by either pipetting up and down or flicking with fingers, NOT vortexing.
- Resuspend the solution in Y*50 μl staining buffer, then keep on ice.
Add antibodies.
- The antibodies are fluorescent. From this point hence, try to keep tubes covered and on ice.
- Prepare X*Y of 1.5 ml test tubes labeled according to antibody and sample.
- Add 50 μl Staining Buffer to each 1.5 ml test tube.
- For each small test tube in a row, add 1 μl of its kind of (primary) antibody to it.
- For each small test tube in a column, add 50 μl of the new cell solution.
- Now the primary antibody will begin binding.
Incubate for 20+ min, wash x2, and resuspend.
- Incubate on ice for 20+ min while protected from light by aluminum foil.
- Wash twice. ((Add 1 ml staining buffer, resuspend, centrifuge (400 G, 5 min, 4 ºC), discard supernatant) x2.)
- Finally, resuspend cells in 400 μl staining buffer.
Analyze with flow cytometer.
- The entire protocol and flow cytometry analysis must be done ASAP and on the same day that tissue was harvested. Results deteriorate quickly with time, even when cells are frozen or fixed with formalin.
- Don't vortex for the same reason as before. Use other means of mixing the contents of each 1.5 ml tube right before analysis.
- When you're graphing the results, consult information on what the x and y axes should be. Don't forget to gate regions on your charts where appropriate.
- Transfer data from the program that comes with the flow cytometer to Word/Excel or any other processor you use.