Labeling cells with monoclonal antibodies (mAbs) for Flow Cytometry
- 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.
- Plate vortexer. If not available, the cells can be mixed with wash
buffers in the 96-well plate using a multichannel or single channel
- Flow cytometer.
- Single and multichannel
- 96-well polystyrene V-bottom plates
- Micropipettor tips
- First wash buffer
|20% NaN3 in PBS
|Horse serum (GIBCO
- Second wash buffer: same as first wash buffer, with horse serum
- 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
- 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. 2% buffered formaldehyde: 25 ml of 37% formaldehyde + 475 ml PBS.
Labeling Cells for use in Single Color Flow Cytometry
- 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.
- 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.
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.
- Add 100 μl of the cell suspension (106
cells) to each well. Incubate on ice for 15 minutes.
- 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.
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
- 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.
- 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.
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
g/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.
- Prepare cells as outlined for single fluorescence labeling.
Record the antibody placement on a 8 x 12 grid sheet. Include second step
reagent only (background fluorescence) controls.
- 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.
- 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
- 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.
- Wash, fix and store the cells as described for single