| A. Common initial procedure when the EuroFlow antibody panel includes SmIg staining |
| If the EuroFlow antibody panel is going to be applied to a sample that includes SmIg staining, follow these initial steps; otherwise go directly to the backbone, surface or intracellular staining protocols (sections B, C, D, respectively): |
|
1. Pipette 300 μl of sample into a 10-ml tube (see Note 1). Note 1: For small samples (i.e. CSF, vitreous aspirates) spin down the total volume (5 min at 540 g), discard the supernatant (see point 5) and resuspend in 300 μl of PBS+0.5% of bovine serum albumin (BSA)+0.09% sodium azide (NaN3).
2. Add 10 ml filtered PBS+0.5% BSA+0.09% NaN3
3. Mix well
4. Centrifuge for 5 min at 540 g
5. Discard the supernatant using a Pasteur pipette or vacuum system without disturbing the cell pellet
6. Add 10 ml PBS+0.5% of BSA+0.09% NaN3 to the cell pellet
7. Mix well
8. Centrifuge for 5 min at 540 g
9. Discard the supernatant using a Pasteur pipette or vacuum system without disturbing the cell pellet
10. Resuspend the cell pellet in 200 μl of PBS+0.5% BSA+0.09% NaN3
11. Continue with conventional EuroFlow SOPs for staining of cell surface or cell surface plus intracellular markers as described below in procedures B, C and D, respectively |
| |
| B. Staining of backbone markers |
|
1. Calculate the total volume of surface membrane backbone antibodies based on the number of tubes in the panel (see Note 2). Note 2: Intracellular backbone markers should not be added here.
2. Pipette these antibodies in one tube (backbone tube)
3. Calculate the total volume of sample to be stained, also based on the number of tubes in the panel and a volume of 50 μl per tube
4. Pipette this sample volume into the backbone tube
5. Mix well
6. Pipette equal amounts of the sample/backbone mix into the various tubes included in the applied EuroFlow panel (see Note 3). Note 3: Both the volume pipetted into each tube and the overall number of tubes depends on the specific EuroFlow panel that is applied.
7. Continue with the steps described below in procedure C
|
| |
| C. Staining of surface markers only (see Note 4): |
|
Note 4: PCD tube 2 is processed identically to PCD tube 1 as described in section D if CD138-PacO is used. |
|
1. Add the appropriate volume of antibodies directed against cell surface markers (except for the backbone markers), as recommended for each specific EuroFlow panel
2. If necessary, use PBS+0.5% BSA+0.09% NaN3 to reach a final volume of 100 μl per tube (see information on the EuroFlow panels)
3. Mix well
4. Incubate for 15 min at room temperature (RT) protected from light
5. Add 2 ml of 1x FACS Lysing Solution (10x FACS Lysing Solution diluted 1/10 vol/vol in distilled water (dH2O))
6. Mix well
7. Incubate for 10 min at RT protected from light
8. Centrifuge for 5 min at 540 g
9. Discard the supernatant using a Pasteur pipette or vacuum system without disturbing the cell pellet, leaving approximately 50 μl residual volume in each tube
10. Add 2 ml of PBS+0.5% BSA+0.09% NaN3 to the cell pellet
11. Mix well
12. Centrifuge for 5 min at 540 g
13. Discard the supernatant using a Pasteur pipette or vacuum system without disturbing the cell pellet, leaving approximately 50 μl residual volume in each tube
14. Resuspend the cell pellet in 200 μl PBS+0.5% BSA+0.09% NaN3
15. Acquire the cells after staining or (if not immediately acquired) store at 4 °C maximally for 3 h until measured in the flow cytometer |
| |
| D. Combined staining of intracellular and surface membrane markers (see Note 5): |
|
Note 5: Tube 4 of the AML/MDS panel should be stained/processed further as described in Procedure E |
|
1. Add the appropriate volumes of antibodies for cell surface markers, as recommended for each specific EuroFlow panel
2. If necessary, use PBS+0.5% BSA+0.09% NaN3 to reach a volume of 100 μl per tube (see information on the EuroFlow panels)
3. Mix well
4. Incubate for 15 min at RT protected from light
5. Add 2 ml of PBS+0.5% BSA+0.09% NaN3 to the cell pellet
6. Mix well
7. Centrifuge for 5 min at 540 g
8. Discard the supernatant using a Pasteur pipette or vacuum system without disturbing the cell pellet, leaving approximately 50 μl residual volume in each tube
9. Resuspend the cell pellet by mixing gently
10. Add 100 μl of Reagent A (fixative; Fix&Perm, An der Grub, Vienna, Austria)
11. Incubate for 15 min at RT protected from light
12. Add 2 ml of PBS+0.5% BSA+0.09% NaN3 to the cell pellet
13. Mix well
14. Centrifuge for 5 min at 540 g
15. Discard the supernatant using a Pasteur pipette or vacuum system without disturbing the cell pellet, leaving approximately 50 μl residual volume in each tube
16. Resuspend the cell pellet by mixing gently
17. Add 100 μl of Reagent B (permeabilizing solution; Fix&Perm)
18. Mix well
19. Add the appropriate volume of the intracellular antibodies (see EuroFlow panels)
20. Mix well
21. Incubate for 15 min at RT protected from light
22. Add 2 ml of PBS+0.5% BSA+0.09% NaN3 to the cell pellet
23. Mix well
24. Centrifuge for 5 min at 540 g
25. Discard the supernatant using a Pasteur pipette or vacuum system without disturbing the cell pellet, leaving approximately 50 μl residual volume in each tube
26. Resuspend the cell pellet in 200 μl PBS+0.5% BSA+0.09% NaN3
27. Acquire the cells after staining or (if not immediately acquired) store at 4 °C maximally for 3 h until measured in the flow cytometer. |
| |
| E. Nuclear (Nu)TdT staining (Tube 4 AML/MDS EuroFlow panel): |
1. Continued from procedure C step 13
2. Add the appropriate amount of the TdT antibody to the cell pellet
3. Mix well
4. Incubate for 15 min at RT protected from light
5. Add 2 ml of PBS+BSA 0.5%+0.09% NaN3 to the cell pellet
6. Mix well
7. Centrifuge for 5 min at 540 g
8. Resuspend the cell pellet in 200 μl PBS+BSA 0.5%+0.09% NaN3
9. Acquire the cells after staining or (if not immediately acquired) store at 4 °C maximally for 3 h until measured in the flow cytometer.
|
| Overview of protocol Sections for the various EuroFlow antibody panels and corresponding tubes. |
