Flow cytometric determination of leukocyte surface antigens in whole blood
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Flow cytometric determination of leukocyte surface antigens in whole blood
This document assumes that you have a mouse IgG monoclonal antibody directed to the antigen you wish to quantitate and that you have fluorescein labeled and fully characterized this antibody.
Quantitation of cell surface antigens in whole blood with the flow cytometer is very simple:
- Collect blood
- Add antibody
- Calibrate the flow cytometer
- Make the measurements
Reagents
- PBS (Phosphate buffered saline) pH 7.4
- Saponin (Sigma) - make 10 mg/ml saponin in PBS. Add azide & store in refrigerator.
- Blood
- Antibody
Blood collection
Collect blood by standard venipuncture into an appropriate anticoagulant. Use EDTA or heparin (10 iu/ml). Do not use citrate, this produces an acidic environment which will quench your fluorescein labeled antibody!Collect blood with a large gauge needle into a syringe. Do not subject the sample to large stresses by pulling too hard on the syringe. If you do this you will certainly damage erythrocytes and probably also damage leukocytes.
You may collect the sample directly into anticoagulant in the syringe. If you collect in a syringe without anticoagulant, carefully introduce the blood into a tube containing anticoagulant. Do not allow the sample to froth in either case.
HOWEVER YOU COLLECT THE BLOOD, ENSURE THAT IT IS COMPLETELY MIXED WITH ANTICOAGULANT. MIX BY INVERSION AT LEAST 10 TIMES!
If you are conducting a study of patient groups it is desirable to draw samples from matched control subjects at the same time. Try to make sure that all blood is drawn at a similar time of day; if not possible, record the time of day it was drawn and use an appropriate control subject.
If you are interested in "resting" levels of antigens, immediately put the tube of blood into ice. KEEP COLD THROUGHOUT.
Add antibody to the blood
Divide the blood into the appropriate aliquots and add labeled mAb to each aliquot as desired. Make sure that the final mAb concentration is at saturation - you may need to determine this by titration. Do blocked samples as necessary.Incubate 30 minutes on ice.
Note that this is for directly labeled antibody. If you need to use an indirect technique, you will need to do washes and incubate with secondary reagent at this point.
Make measurements
Prepare a stock solution of saponin, 10 mg/ml in PBS. Add 0.1% sodium azide and store in the refrigerator.Before the measurement phase of the experiment starts, prepare a sufficient quantity of tubes each containing 0.05 ml saponin solution. Place these on ice and allow to cool.
Add 0.025 ml blood to a 0.05 ml aliquot of saponin and mix with the pipette tip. Wait 5 seconds then add 0.45 ml PBS. Periodically agitate for a short period, up to 30 seconds, monitoring sample clarity visually. It is easy to tell when the erythrocytes lyse, because the sample clears rapidly. As soon as lysis has occurred, return the tube to ice. Allow a further 10 seconds to complete the lysis and read on the flow cytometer. Use characteristic forward versus orthogonal light scattering to set a gate for the type of cells in which you are interested. Acquire at least 5000 events and store pending off-line analysis.
You can add up to 0.05 ml blood to each saponin aliquot with good results. Do not add more, otherwise the erythrocytes will not lyse properly.
It seems that human leukocytes are quite resistant to saponin lysis, and the samples will be stable on ice for about 3-5 minutes. Other species differ in this respect and you may need to modify the lysis protocol to suit. I have had good results with human, rabbit, rat, baboon and pig (but not hamster). For optimum leukocyte stability it may be necessary to keep the sample cold while it is running on the cytometer (use an ice bath).
The concentration of saponin used here does not appear to remove antigens from the cell membrane, however test this with purified cells if you are suspicious.
