MONOCLONAL ANTIBODY STAINING PROCEDURE FOR WHOLE BLOOD USING NH4CL FOR LYSIS OF RED BLOOD CELLS
I. SAMPLE
A) Blood drawn into EDTA vacutainer tubes, keep at room temperature and process within 24 hours.
B) Blood drawn into Heparin vacutainer tubes, keep at room temperature and process within 24 hours.
II. REAGENTS
A) Antibodies
1. Primary antibodies: usually purchased or your own monoclonals
a) If these are conjugated with a fluorochrome (i.e., FITC, PE, or other) use the DIRECT STAINING PROCEDURE.
b) If the primary antibody is not fluorescence-conjugated, use the INDIRECT STAINING PROCEDURE.
2. Secondary antibodies: fluoresceinated polyclonal antibody
B) Buffer: PBS (e.g., Ca 2+ and Mg2+ free, Irvine Scientific, cat. #9240) + 2% newborn calf serum (or 0.2% BSA) + 0.1% sodium azide.
C) NH4Cl Lysing Solution (see attached recipe)
III. PROCEDURE
DIRECT STAINING
1. First, add 50 microliters of BUFFER, then the appropriate amount of monoclonal antibody to the bottom of tubes.
Note: For dual- or multi-color staining, add all your directly-conjugated antibodies at the same time.
2. Add 50 microliters of whole blood to the bottom of the tubes.
3. Vortex briefly and incubate 15 minutes at room temperature.
4. Wash twice with ~1ml of buffer; centrifuge at 250g for 5 minutes.
5. Take off as much buffer as possible without disturbing the pellet.
6. Vortex briefly and add 1 ml room temperature 1 x NH4 CL lysing solution to the pellet.
7. Vortex immediately and incubate samples between 7 and 10 minutes at room temperature protected from light.
Note: Do not exceed this time.
8. Spin down samples for 5 minutes at 200 g.
9. Take off lysing solution as completely as possible.
10. Resuspend samples in ~ 0.5 ml of buffer and hold in refrigerator (or on ice) protected from light prior to analysis.
INDIRECT STAINING
1-4. Process cell samples as above using a working dilution of unlabeled monoclonal antibody.
5. Resuspend cell pellet in 100 microliters of working dilution of the fluoresceinated second antibody.
6. Vortex briefly and incubate for 20 minutes at room temperature.
7. Wash twice with ~1ml of buffer; centrifuge at 250g for 5 minutes.
8. Process samples as described above in 5-10 of DIRECT STAINING procedure.
Note: If you increase the amount of whole blood you stain you have to increase the amount of lysing buffer to maintain a ratio of 1:20 of blood to lysing solution.
AMMONIUM CHLORIDE LYSING SOLUTION – 10X
Reagents Amount:
Ammonium Chloride, ACS 82.9 g
Potassium Bicarbonate, USP 10.0 g
Ethylenediamine tetraacetic acid (EDTA) disodium salt 0.37 g
Water, glass distilled qsad 1.0 liter
Adjust pH to 7.2 and keep in tightly closed container at 4°C. To prepare a 1X working solution (to be used at room temperature), dilute 10X 1:10 with glass distilled water. Keep tightly closed and discard at the end of the day.
A) Blood drawn into EDTA vacutainer tubes, keep at room temperature and process within 24 hours.
B) Blood drawn into Heparin vacutainer tubes, keep at room temperature and process within 24 hours.
II. REAGENTS
A) Antibodies
1. Primary antibodies: usually purchased or your own monoclonals
a) If these are conjugated with a fluorochrome (i.e., FITC, PE, or other) use the DIRECT STAINING PROCEDURE.
b) If the primary antibody is not fluorescence-conjugated, use the INDIRECT STAINING PROCEDURE.
2. Secondary antibodies: fluoresceinated polyclonal antibody
B) Buffer: PBS (e.g., Ca 2+ and Mg2+ free, Irvine Scientific, cat. #9240) + 2% newborn calf serum (or 0.2% BSA) + 0.1% sodium azide.
C) NH4Cl Lysing Solution (see attached recipe)
III. PROCEDURE
DIRECT STAINING
1. First, add 50 microliters of BUFFER, then the appropriate amount of monoclonal antibody to the bottom of tubes.
Note: For dual- or multi-color staining, add all your directly-conjugated antibodies at the same time.
2. Add 50 microliters of whole blood to the bottom of the tubes.
3. Vortex briefly and incubate 15 minutes at room temperature.
4. Wash twice with ~1ml of buffer; centrifuge at 250g for 5 minutes.
5. Take off as much buffer as possible without disturbing the pellet.
6. Vortex briefly and add 1 ml room temperature 1 x NH4 CL lysing solution to the pellet.
7. Vortex immediately and incubate samples between 7 and 10 minutes at room temperature protected from light.
Note: Do not exceed this time.
8. Spin down samples for 5 minutes at 200 g.
9. Take off lysing solution as completely as possible.
10. Resuspend samples in ~ 0.5 ml of buffer and hold in refrigerator (or on ice) protected from light prior to analysis.
INDIRECT STAINING
1-4. Process cell samples as above using a working dilution of unlabeled monoclonal antibody.
5. Resuspend cell pellet in 100 microliters of working dilution of the fluoresceinated second antibody.
6. Vortex briefly and incubate for 20 minutes at room temperature.
7. Wash twice with ~1ml of buffer; centrifuge at 250g for 5 minutes.
8. Process samples as described above in 5-10 of DIRECT STAINING procedure.
Note: If you increase the amount of whole blood you stain you have to increase the amount of lysing buffer to maintain a ratio of 1:20 of blood to lysing solution.
AMMONIUM CHLORIDE LYSING SOLUTION – 10X
Reagents Amount:
Ammonium Chloride, ACS 82.9 g
Potassium Bicarbonate, USP 10.0 g
Ethylenediamine tetraacetic acid (EDTA) disodium salt 0.37 g
Water, glass distilled qsad 1.0 liter
Adjust pH to 7.2 and keep in tightly closed container at 4°C. To prepare a 1X working solution (to be used at room temperature), dilute 10X 1:10 with glass distilled water. Keep tightly closed and discard at the end of the day.
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