Titrating fluorescently-labelled anti-human antibodies for flow cytometry using whole blood samples in deep well plates

Purpose

Fluorophore-labeled antibodies--especially those produced in the laboratory and not purchased from a commercial supplier--must be "titered" to obtain the optimal concentration to be used for staining. Normally, when using PBMC or splenocyte samples, this is a trivial procedure, not requiring a detailed protocol. However, when performing a titration experiment with whole blood samples, there are liquid handling issues that are non-obvious, providing the motivation for this protocol. For convenience and to reduce potential errors, this protocol uses 96 well deep well plates (2 ml/well).

Contents of this document

Crucial parameters

Keep the blood volume to 100 microliters. This will allow you to add 1 ml of FACS lyse, a volume that is easily accomodated by the deep well plates.

Perform at least 8 three-fold serial dilutions, starting at an antibody concentration in the first stain of approximately 10 micrograms/ml.

If you will be acquiring samples on the Multiwell Autosampler, you must use plates that are compatible with the instrument. A complete listing of these plates is found in the document BD MULTIWELL™ AUTOSAMPLER PLATE DEFINITION GUIDE, and in the materials section below. If you are acquiring samples on the FACS Aria, we recommend transferring the samples to 1.2 ml polypropylene cluster tubes, which fit in a standard 96-well rack with spacing that matches the 96 well plate. Finally, if you are acquiring the samples on the FACS Calibur, you should transfer the samples to standard 12 x 75 FACS tubes.

Materials

• Impact2 electronic multichannel pipettor from Matrix Technologies.
• 1250 ul tips for the Impact2. (cat# 8051 for non-sterile, non-barrier $56/case; cat#8052 for sterile, non-barrier $68/case; cat#8056 for ecotip refills $49/case; and cat#8055 for sterile, barrier tips $91/case; cases sizes are 6 racks with 120 tips per rack). Note that these catalog numbers and configurations may soon change (fall 2003?).
• Aspiration manifold from VP Scientific with 35 mm long prongs (cat# VP 187A). At each end of the manifold, place a piece of cut 2 mm thick tygon tubing to prevent the manifold from reaching the bottom of the plate.
• BD FALCON 96-Well DeepWell Polypropylene Plates, 2 ml/well (VWR cat# 62406-496) Note that these are required for use of the Multiwell Autosampler on the FACS Calibur.
• FACS Lysing solution (Becton Dickinson 349202)
• FACS wash
• Formaldehyde, 10%, methanol free, Ultra Pure[50-00-0] (Polysciences  04018-1)
• Cluster tubes in racks (non-sterile: VWR 29442-606; bulk non-sterile: VWR 29442-602). These can be used on the FACS Aria. If you are using labels that can be read on the FACS Calibur, then these tubes are not required.
• Titer Plate Shaker, Barnstead/Lab-Line (VWR 57019-600), or conventional vortex mixer with a disk head (e.g., the hockey puck).

Protocol

1. Add 27 ul of FACS buffer to the each well in the first row of the deep well plate. Each mAb to be titrated will be in a separate column (see diagram at step 5 below). Use as many columns as you have antibodies to titrate. You should add an extra column for unstained samples (though you'll only need 1-2 unstained samples). You should be careful to pipette the buffer directly to the bottom of the well, and to avoid leaving drops on the side of the well. This also applies to steps 2, 3, and 4. AVOID CREATING BUBBLES at all pipetting steps.
2. Add 20 ul of FACS buffer to each well in rows 2-8.
3. Add 3 ul of the concentrated mAb stock to a well in a corresponding column in the first row of the plate. The concentrated mAb stocks will most likely vary in concentration, but should usually be around 2 mg/ml.
4. With a single-channel P-20 pipetter set at 10 ul, carefully mix the contents of row-1/column-1, and then perform serial dilutions in subsequent rows in column 1. Withdraw the final 10 ul of the solution from row 8, leaving behind 20 ul. Use a separate columne for unstained samples. AVOID CREATING BUBBLES by not depressing the button on the pipetter completely during the mixing process. It is also possible to perform the serial dilutions in multiple columns at the same time, if you have a multichannel pipetter that will reliably pipette 10 microliters. On the day that I wrote this protocol, ours wouldn't do this.
5. Repeat for additional columns.
   
   
6. Add 100 ul of whole blood to each well. This could be done with a multichannel pipetter, with a single channel pipetter, or with a repeater pipette (I would probably use the latter). If you use a repeater pipette, mix the contents of the plate on the plate shaker. Start at a slow speed an increase to ensure mixing without splashing between wells.
7. Incubate (15 minutes at room temperature in a drawer).
8. With the programmable Impact2 electronic multichannel pipetter, add 1000 ul of FACS lyse to each well and mix. The basic program for the Impact 2 is (1) Fill 1000 ul; (2) dispense 1000 ul; (3) mix 500 ul.
9. Incubate for 10-15 minutes at room temperature. Keep the samples dark by placing the plate in a drawer, or wrap in foil.
10. Spin (??? g)
11. Aspirate supes with the manifold. Aspiration with the manifold will leave behind between 100-400 ul of buffer. This is might be more than we'd like for washing, but on the other hand, aspiration with longer prongs would risk aspirating the cells right out of the plate.
    
    Note that we are not comfortable with simply dumping the contents of the plate into the sink (or a bucket of bleach), due possible cross-contamination of samples. We are still evaluating this stage of the protocol. In particular, it might be possible to combine aspiration with subsequent flicking. On the other hand, we don't seem to need to do this.
12. Mix on plate shaker to resuspend the pellet. Alternatively, it might be necessary to mix the samples on a conventional vortex mixer with a disk-type head. The point is to disrupt the cell pellets, but they are difficult to see in these plates. We are still evaluating this step.
13. Wash 2-3 times with 1000 ul FACS buffer, centrifuging the samples and aspirating the supernatants with the manifold at each step. Use the IMPACT2 for dispensing liquid at this step. If you are careful not to touch the wells with the tips, I don't think it is necessary to change the tips for each row. After adding FACS buffer, it is possible to mix the samples on the plate shaker, if you are careful to start the shaker at a slow speed and then you slowly increase the speed to the point well below that at which the samples would spill from one well to another. We don't know if this is necessary.
14. After the final aspiration of the wash buffer, you have several options, depending upon which instrument you will be using to acquire your samples. If you are usign the Multiwell Autosampler, you can simply add a volume of 2% formaldehyde equal to the residual volume after aspiration ("1 volume") to each of the wells and mix. If you are using the FACS Aria, you should prepare cluster tubes that have "1 volume" of 2% formaldehyde each, and then, using a multichannel pipetter, mix the residual volume of cells and transfer to cluster tubes racked in the same orientation as the plate. If you are using FACS tubes, you should probably transfer the samples individually. You could also use the Impact2, which has adjustable spacing between the channnels, to transfer to racked 12 x 75 FACS tubes.