Flow Cytometry Laboratory

Labeling cells with monoclonal antibodies (mAbs) for Flow Cytometry

Equipment

1. Refrigerated centrifuge which will accept 96-well plate carriers, capable of spinning 2000 RPM. If a centrifuge which will accept plate carriers is unavailable, the procedure can be carried out in 12 x 75 mm polypropylene tubes or microcentrifuge tubes, using aspiration to remove supernates. It is important that the cells and reagents remain chilled at all times, however.
2. Plate vortexer. If not available, the cells can be mixed with wash buffers in the 96-well plate using a multichannel or single channel micropipettor.
3. Flow cytometer.
4. Single and multichannel

Supplies

1. 96-well polystyrene V-bottom plates
2. Micropipettor tips
   

Solutions

1. First wash buffer

PBS	450 ml
Acid-citrate-dextrose	50 ml
20% NaN3 in PBS	5 ml
Horse serum (GIBCO BRL)	10 ml

2. Second wash buffer: same as first wash buffer, with horse serum omitted.
3. 3. Primary monoclonal antibodies: Working stocks of 15 μg/ml (in .09% azide) in first wash buffer are kept refrigerated. Working stocks should be maintained free of particulates by filtration through a 0.22μ filter.
4. 4. Second step reagents (2nd) : Anti-mouse immunoglobulin antibodies conjugated to a fluorescent compound such as fluorescein isothiocyanate (FITC), Alexa fluor 488 or 647, phycoerythrin (PE), PE-Cy5.5, and Cy5. Working dilutions depend upon the source of the antibody. They need to be titrated before use. Dilutions vary with the fluorochrome used and the commercial source of the reagent. Working dilutions may vary from 1:100 to 1:4,000. Conjugated antibodies are readily available from several commercial sources. We routinely use the products supplied by Invitrogen and Southern Biotechnology Associates (available through Fisher Scientific in the US).
5. 5. 2% buffered formaldehyde: 25 ml of 37% formaldehyde + 475 ml PBS.

Labeling Cells for use in Single Color Flow Cytometry

1. All cells and reagents must be maintained at 4oC on ice at all times. Make sure the centrifuge and plate carriers are cold. The pH of the buffers should be 7.2 - 7.4.
2. Prepare lymphocytes or other cells by Hypaque-Ficoll separation, as buffy coat suspension, or as whole blood lysate (or as protocol dictates for the experiment). Cells should be washed several times to remove platelets. After the final wash, resuspend the cells in first wash buffer at a concentration of 107 cells/ml and place on ice.
3. Record the antibody placement on a 8 x 12 grid sheet. Set up a control well containing cells, first wash buffer and second step reagent(s) only (control for non-specific binding). Include an irrelevant monoclonal antibody of each isotype used as an isotype control. Include all data, i.e. cell type, treatment of cells, cell count, type and dilution of second step reagent, and incubation times. Place a v-bottom 96-well plate on ice and add 50 μl of monoclonal antibodies (15 μg/ml) to the appropriate wells.
4. Add 100 μl of the cell suspension (106 cells) to each well. Incubate on ice for 15 minutes.
5. Centrifuge the plate for 3 minutes at 2000 RPM at 4oC. Remove supernatant by flicking the plate. Move quickly to avoid warming the cells. Vibrate the plate briefly on the plate vortexer to loosen the cell pellets. Add 200 μl of first wash buffer to the pellets. Check by looking through the plate from the bottom to be sure the cells are evenly dispersed. Repeat step 5 three times for a total of three complete washes.
6. After the last wash, add 100 μl of appropriately diluted 2nd reagent to all wells, including the control well that contains cells but no primary mAbs. Check again to see that the cells are evenly dispersed. If necessary, resuspend the cells using a multichannel micropipettor. Incubate 15 minutes on ice in the dark.
7. After incubation, wash 2 times as above, using second wash buffer. Resuspend the cells in 200 μl 2% PBS buffered formaldehyde. Labeled cells can be examined live immediately after washing also. Fixation is just a way of preserving the cells for later analysis.
8. Seal the plate with Parafilm and store the cells refrigerated in the dark. Fluorochromes will be quenched if exposed to fluorescent light for long periods of time. The cells may be examined immediately or after a delay of up to two weeks.
9. To examine the cells, move the contents of each well to a 12 x 75 mm tube

Labeling Cells for use in Multicolor Color Flow Cytometry

Cells can be simultaneously examined for expression of up to 4 different molecules, as long as the mAbs used are of different isotype. Isotype-specific 2nd step antibodies conjugated to 4 different fluorochromes can be used to visualize the pattern of expression of two or more molecules expressed on the same or different cell subsets. Multicolor labeling provides a way to analyze the composition of complex populations of leukocytes. It also provides a way to minimize the number of samples needed for analysis. The protocol for labeling is the same as for single flow cytometry. At present, there are few directly conjugated 2nd step reagents for use in species other than humans and laboratory animal species so indirect labeling is necessary. Each mAb used in indirect multicolor flow cytometry must be of a different isotype. For multicolor analysis, it is convenient to prepare cocktails of mAbs so that all antibodies are present at 15 mg/ml in a single solution. This speeds up the process of making up labeling plates, and also keeps the volume in each well from getting too large. Cocktails of 2nd step reagents can be prepared also at the time of use.

Equipment, supplies and solutions are the same as indicated for single fluorescence labeling. The additional reagents needed are isotype-specific anti-mouse immunoglobulin antibodies conjugated to different fluorochromes, typically FITC and PE. Isotype-specific reagents are available from Southern Biotechnology, as well as other commercial sources.

Procedure

1. Prepare cells as outlined for single fluorescence labeling.
2. Record the antibody placement on a 8 x 12 grid sheet. Include second step reagent only (background fluorescence) controls.
3. Place a v-bottom 96-well plate on ice and add monoclonal antibodies to the wells as indicated on the grid sheet. Add 50 μl of each mAb or cocktail of mAbs to each well.
4. Add 100 μl of the cell suspension (106 cells) to each well. Check to make sure there was adequate mixing. Incubate for 15 minutes on ice. Centrifuge and wash three times as indicated in the single fluorescence procedure.
5. Make up the appropriate volumes of 2nd step reagents and add 25 or 50 μl to each well. Mix with a multi-channel pipettor. Incubate 15 minutes on ice in the dark.
6. Wash, fix and store the cells as described for single fluorescence

 

Updated 2/26/09