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Titrate Your Antibodies

Directly Labeled:

1. Determine concentration of Antibody (Ab) and centrifuge 10 min at 15,000 X g, 4° C to remove aggregates. Discard pellet.
2. Start with Ab stock at 300 µg/ml in Phosphate Buffered Saline (PBS). Serial dilute six tubes:

10 µl Ab + 20 µl PBS (or 20 µl of previous dilution) = 30 µl

3. Resuspend cells in 200 µg/ml normal IgG of detecting species at 5 - 10 X 10⁶ cells/ml.
4. Aliquot 50 µl of cells in (Falcon #2054) 12 X 75 mm tubes on ice.
5. Add 10 µl of each Ab dilution. Also prepare a tube with cells alone (autofluorescence control) and one with the detecting species IgG negative control.
6. Gently mix and incubate 15 - 45 min on ice IN THE DARK.
7. Add 2 ml cold Washing Buffer (e.g. PBS + 2% Bovine Serum Albumin), gently mix and centrifuge 5 min at 200 X g, 4° C.
8. Aspirate and discard supernatant. Gently vortex to break up cell pellet and repeat Step 7.
9. Resuspend pellet in 100 µl protein-free, cold PBS and mix well.
10. Add 300 µl cold PBS + 2% paraformaldehyde. Store in refrigerator, light protected for up to 10 days. Can skip fixation and use Propidium Iodide at 0.5 µg/ml in Washing Buffer and run within 4 hours.

Indirectly Labeled:

1. Prepare cells in 200 µg/ml normal IgG of the second antibody species (e.g. goat) at 5 - 10 X 10⁶ cells/ml.
2. Start with Ab stock at 300 µg/ml in PBS. Serial dilute six tubes:

       10 µl Ab + 20 µl PBS (or 20 µl of previous dilution) = 30 µl

3. Aliquot 50 µl of cells in (Falcon #2054) 12 X 75 mm tubes on ice.
4. Add 10 µl of each Ab dilution. Also prepare a tube with cells alone (autofluorescence control) and isotype negative control.
5. Gently mix incubate 15 - 45 min on ice.
6. Add 2 ml cold Washing Buffer (e.g. PBS + 2% BSA), gently mix and centrifuge 5 min at 200 X g, 4° C.
7. Aspirate and discard supernatant. Gently vortex to break up cell pellet and repeat Step 6.
8. Resuspend cells in 100 µl of appropriately diluted fluorochrome-conjugated second antibody (e.g. goat anti-mouse FITC).
9. Incubate 15 - 45 min on ice IN THE DARK.
10. Wash twice as above (Steps 6 & 7).
11. Resuspend pellet in 100 µl protein-free, cold PBS and mix well.
12. Add 300 µl cold PBS + 2% paraformaldehyde. Store in refrigerator, light protected for up to 10 days. Can skip fixation and use Propidium Iodide at 0.5 µg/ml in Washing Buffer and run within 4 hours.

Here's an example of a titration showing the expected decrease in fluorescence with a corresponding increase in the signal to noise ratio:

Reference

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Dennis J. Young, UCSD Cancer Center Flow Cytometry Core Facility