Flow Cytometry

FLOW CYTOMETRY PROTOCOL

BLOOD SAMPLE PREPARATION

• For each 1 ml of blood, add 14 ml of room temperature FCM Lysing solution to lyse the red blood cells. The cells will not lyse correctly if the solution is cold.
• Incubate for 5 minutes at room temperature. Do not exceed 5 minutes, as the white blood cells will begin to lyse beyond 5 minutes.
• Centrifuge for 5 minutes at 1000 RPM for human blood.
• Carefully aspirate supernatant, then re-suspend the pellet in approximately 50 ml cold 1X PBS. Take a small sample to perform a cell count.
• Centrifuge for 5 minutes at 1000 RPM for human blood.
• Aspirate supernatant.

STAIN PREPARATION
Fix cells or prepare live cells for staining.
NOTE: It is very important to block Fc receptors for certain cell types.
• Once supernatant is aspirated from cell preparation, re-suspend pellet in enough 1X PBS to have a final cell concentration of 10 million cells/ml.
• Block by incubating the cell suspension with 1 mg of goat serum per 1 ml of cell suspension for 10 minutes. Do not rinse. Proceed with staining.

DIRECT STAINING
• Label tubes.
• Add 20 ?l of fluorochrome-conjugated antibodies to tubes.
• Add 100 ?l of the prepared cell suspension (equal to 1 million cells) to each tube.
• Vortex and incubate for 15-30 minutes in a covered ice bucket.
• To wash off excess antibody following staining, add 1.5-2 ml of 1X PBS to each tube.
• Centrifuge in tabletop microfuge for 5 minutes at 2000 RPM. This speed should be increased to 3000 or 4000 RPM for intracellular staining.
• Aspirate supernatant, being careful not to disturb pellet.
• Resuspend pellets in 500 ?l of 1% paraformaldehyde. Tubes can be stored in the dark for 24 hours (maximum for intracellular staining) to 1 week (maximum for surface staining).

INDIRECT STAINING
• Label tubes.
• Add unconjugated primary antibodies to tubes. Use approximately 1 ?g per tube.
• Add 100 ?l of the prepared cell suspension (equal to 1 million cells) to each tube.
• Vortex and incubate for 15-30 min in a covered ice bucket.
• To wash off excess antibody following staining, add 1.5-2 ml of 1X PBS to each tube.
• Centrifuge in tabletop microfuge for 5 minutes at 2000 RPM (or 3000-4000 RPM for intracellular staining).
• Aspirate supernatant, being careful not to disturb pellet.
• Add 100 ?l of 1X PBS to each tube. Add fluorochrome-conjugated secondary antibodies to tubes. Use 0.5-1 ?g of antibody.
• Vortex and incubate for 15-30 minutes in a covered ice bucket.
• To wash off excess antibody following staining, add 1.5-2 ml of 1X PBS to each tube.
• Centrifuge in tabletop microfuge for 5 minutes at 2000 RPM (or 3000-4000 RPM for intracellular staining).
• Aspirate supernatant, being careful not to disturb pellet.
• Resuspend pellets in 500 ?l of 1% paraformaldehyde. Tubes can be stored in the dark for 24 hours (maximum for intracellular staining) to 1 week (maximum for surface staining).

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