FACS in 96-well Plate Protocol
This protocol is used specifically for mice at the Jackson Laboratory, Bar Harbor, Maine. The references to our Flow Cytometry Service, including antibody dilutions, machines used (FACScan and FACSCalibur) and source of Propidium Iodide are site specific. Hopefully, you can adapt this to whatever resources are available at your institute without too much trouble. Because this method uses small volumes of liquid (200 ul compared to 2-4 ml which would be used if treating individual samples in separate tubes) a larger percentage of the red blood cells survive the lysis steps. This may make this preparation method unsuitable for looking for some subtle phenotype differences. I use it successfully for all of my Flow Cytometry work, which mostly involves genotyping.
1. Collect ~ 70 ul blood (in mice this is most efficiently done by periorbital bleeding) using a Heparanized capillary tube into 150 ul Saline EDTA in a 96-well plate (I like to use Rainin R96-OAPV-1CO v-bottom plates with Costar 3931 corner notch lids).
2. Spin plate at 2000 rpm for 2 minutes in a centrifuge1.
3. Remove supernatant by multichannel pipetter set to 160 ul (carefully from side of well so as not to suck up the cell pellet).
4. Add 160 ul 1X ACK Lysis Buffer to wells and mix cells in buffer by pipetting up and down with a multi-channel pipetter.
5. Incubate at 4C for 10 minutes (either on ice or in refrigerator).
6. Spin plate at 2000 rpm for 7 minutes.
7. Remove supernatant by vacuum aspiration using a tabletop vacuum source and 8-channel head. (Alternately, if you choose to aspirate using a multichannel pipetter as in steps 3, then centrifuge step 6 only needs to be for 2 minutes.)
8. Add 200 ul 1X ACK Lysis Buffer to wells and mix cells in buffer by pipetting up and down with a multi-channel pipetter.
9. Incubate at 4C for 10 minutes (either on ice or in refrigerator).
10. Spin plate at 2000 rpm for 2 minutes.
11. 'Flick’ plate over sink to remove supernadent, then dab gently on paper towel to remove any residual.
12. Add 200 ul Counting Solution (PBS with 1% Bovine Serum Albumin (BSA)).
13. Spin plate at 2000 rpm for 2 minutes.
14. Flick’ plate over sink to remove supernadent, then dab gently on paper towel to remove any residual.
15. Vortex plate.
16. Add fluorescently labeled antibodies to stain cells (typically 10 ul per well at dilution recommended by Flow Cytometry Service. Incubate at 4C in dark for 30 minutes (usually by placing in refrigerator).
17. Add 200 ul Counting Solution (PBS with 1% BSA) per well.
18. Spin plate at 2000 rpm for 2 minutes.
19. 'Flick’ plate over sink to remove supernatant, then dab gently on paper towel to remove any residual.
20. Vortex plate.
21. Add 200 ul Counting Solution (with 10% Propidium Iodide Solution from Flow Cytometry Service if you desire live/dead cell discrimination).
22. Take to FACScan or FACSCalibur for analysis (I carry a pipetter, tips and 4 ml tubes with me to Flow Lab, and transfer the cells from plate to tube there. If you are organized, this can eliminate the need to label many individual tubes).
1 We use a Sorvall RT 6000B with swinging buckets. (Note: This speed in this centrifuge with the appropriate plate carriers is very near the limit at which plates crack. If you have problems with excessive plate cracking, I suggest experimenting with reducing centrifuge speed by 100 RPM increments. The appropriate speed will likely still crack plate edges (mine does) but will not crack wells.)
Saline EDTA (500 ml)
4.5 g NaCl (0.9% weight/volume)
2 ml of 0.5 M EDTA (2mM final concentration)
500 ml 18MOhm H2O
Autoclave, store at 4oC.
1x ACK Lysis Buffer (2 liters)
16.58g NH4Cl
2.00g KHCO3
.074g Na2EDTA
pH to 7.2, volume to 2 liters in 18MOhm H2O
Autoclave, store at 4oC.
