Flow Cytometry

PROTOCOL
Santa Cruz Biotechnology, Inc.

A. SAMPLE PREPARATION B. CELL STIMULATION

Prepare cells according to cell type. Stimulate cells as necessary.

Blood (Human, Mouse or Rat) C. STAIN PREPARATION

• For each 1 ml of blood, add 14 ml of room temperature FCM Lysing solu- Fix cells or prepare live cells for staining.
tion (sc-3621) to lyse the red blood cells. The cells will not lyse correctly if
NOTE: It may be desired to block Fc receptors for certain cell types including,
the solution is cold. but not limited to, mouse and rat blood, mouse spleen, mouse bone marrow,
• Incubate for 5 minutes at room temperature on a rotator. Do not exceed etc. A blocking reagent contains a high concentration of immunoglobulin that
5 minutes, as the white blood cells will begin to lyse beyond 5 minutes. will bind to the Fc-receptors on cells. The blocking reagent should ideally be
immunoglobulin from the species whose cells you are staining. Recommended
• Centrifuge for 5 minutes at 1000 RPM for human blood, 2000 RPM for
blocking reagent: 1 µg normal immunoglobulins per 1 million cells.
mouse or rat blood.
• Carefully aspirate supernatant, then resuspend pellet in approximately Live Staining
50 ml cold 1X PBS. Take a small sample to perform a cell count. • Once supernatant is aspirated from cell preparation, resuspend pellet in
enough 1X PBS to have a final cell concentration of 10 million cells/ml.
• Centrifuge for 5 minutes at 1000 RPM for human blood, 2000 RPM for
mouse or rat blood. • Block by incubating the cell suspension with blocking reagent for
10 minutes. Do not rinse. Proceed with staining.
• Aspirate supernatant.
Fixed and Permeabilized Cells for Intracellular Staining
Mouse Spleen or Other Tissue
• Once supernatant is aspirated from cell preparation, resuspend pellet in
• Harvest organ or tissue and prepare single cell suspension.
enough 1X PBS to have a final cell concentration of 10 million cells/ml.
• Pass cell suspension through a 70 µM cell strainer.
• Block by incubating the cell suspension with blocking reagent for
• Centrifuge for 5 minutes at 1000 RPM. 10 minutes.
• Discard supernatant and add 5 ml of room temperature FCM Lysing • Resuspend pellet in approximately 50 ml 1X PBS to wash away any excess
solution (sc-3621). blocking antibody.
• Incubate for 2–3 minutes at room temperature, allowing larger pieces to • Centrifuge for 5 minutes at 1000 RPM.
fall to the bottom of the tube.
• Once supernatant is aspirated from cell preparation, resuspend pellet in
• Carefully pipette the suspension out and deposit into a clean tube. Take a FCM Fixation Buffer (sc-3622). Use 1 ml per million cells.
small sample to perform a cell count.
• Incubate for 30 minutes at room temperature on a rotator.
• Centrifuge for 5 minutes at 1000 RPM.
• Centrifuge for 5 minutes at 1500–2000 RPM. Cells get more buoyant
• Aspirate supernatant. after fixation. If pellet is too small, spin again at a higher RPM, but do not
exceed 3000 RPM.
Suspension Cell Line
• Pipette off cells, rinsing plate to ensure maximum recovery. Take a small • Pour off supernatant. Cells may be lost if aspirating from this point on,
sample to perform a cell count. so always decant. Use a quick motion and don’t allow the supernatant to
wash back and forth over the cells.
• Centrifuge for 5 minutes at 1000 RPM.
• Resuspend pellet in approximately 50 ml 1X PBS to wash away any excess
• Aspirate supernatant. Fixation Buffer.
Monolayer / Adherent Cell Line • Centrifuge for 5 minutes at 1500–2000 RPM.
• Vacuum off media. Rinse plate once with 1X PBS. Vacuum off PBS. • Decant supernatant. At this point, cells can be resuspended in a small
• Add approximately 5 ml of 0.2% EDTA (in PBS) to plate. Using a Trypsin/ amount of PBS and stored for up to 1 month at 4° C. To permeabilize at
EDTA solution in the place of 0.2% EDTA may compromise any cell surface this time, proceed to next step.
staining. NOTE: You should only proceed with permeabilization if you can stain imme-
• Wait for cells to “round up.” Placing the cells in an incubator may speed diately afterwards.
up this process. Check the plate(s) every 5 minutes. • If cells have been stored in PBS, centrifuge for 5 minutes at 1500–2000 RPM
• Add approximately 5 ml of media to neutralize EDTA. and decant supernatant.
• Pipette off cells, rinsing plate to ensure maximum recovery. Take a small • Break up cell pellet and dropwise add the same amount of COLD (stored at
sample to perform a cell count. -20° C) FCM Permeabilization Buffer (sc-3623) at 1 ml per 1 million cells.
Vortex while adding.
• Centrifuge for 5 minutes at 1000 RPM.
• Incubate for 5 minutes only at RT on a rotator.
• Aspirate supernatant.
• Immediately centrifuge for 5 minutes at 2000–2500 RPM. Cells are more
buoyant after permeabilization and much care must be excercised to
maintain volume of cells.

Santa Cruz Biotechnology, Inc. 1.800.457.3801 831.457.3800 fax 831.457.3801 Europe +00800 4573 8000 49 6221 4503 0 www.scbt.com
NOTE: Important: If a pellet is not recovered at this step, be sure to spin
again and try to recover more cells.
• Decant supernatant and add approximately 50 ml 1X PBS to wash away any
excess Permeabilization Buffer.
• Centrifuge for 5 minuntes at 2000–2500 RPM.
• Decant supernatant and resuspend pellet in enough FCM Wash Buffer
(sc-3624), for a final cell concentration of 10 million cells/ml. In the staining
steps, use FCM Wash Buffer in place of 1X PBS.

D. STAINING
Follow protocol for direct or indirect staining.

DIRECT STAINING
(With Fluorochrome-Conjugated Antibodies)
• Label tubes.
• Add 1 µg of fluorochrome-conjugated antibodies to tubes.
• Add 100 µl 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 µl 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
(With Unconjugated Primary Antibodies and Fluorochrome-
Conjugated Mouse IgG Binding Proteins or Secondary Antibodies)
• Label tubes.
• Add unconjugated primary antibodies to tubes. Use approximately 1 µg per
tube.
• Add 100 µl 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 µl of 1X PBS to each tube. Add fluorochrome-conjugated Mouse
IgG Binding Proteins or secondary antibodies to tubes. Use 0.5–1 µg of
binding protein/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 µl of 1% paraformaldehyde. Tubes can be stored
in the dark for 24 hours (maximum for intracellular staining) to 1 week
(maximum for surface staining).

E. ACQUIRE
Acquire within 24 hours.

Santa Cruz Biotechnology, Inc. 1.800.457.3801 831.457.3800 fax 831.457.3801 Europe +00800 4573 8000 49 6221 4503 0 www.scbt.com