(a) Cells stored in liquid nitrogen

1. Prepare PBS/BSA buffer (phosphate buffered saline pH 7.4 and 1% BSA).
2. Carefully remove cells from liquid nitrogen storage.
3. Thaw rapidly using PBS/BSA buffer and place into a 15 ml conical centrifuge tube.
4. Centrifuge at 400 g for 5 minutes.
5. Discard supernatant and resuspend pellet in an appropriate amount of
6. PBS/BSA buffer.

(b) Tissue culture cell lines in suspension

1. Prepare PBS/BSA buffer (phosphate buffered saline pH 7.4 and 1% BSA).
2. Decant cells from tissue culture flask into 15 ml conical centrifuge tube(s).
3. Centrifuge at 400 g for 5 minutes.
4. Discard supernatant and resuspend pellet in 10 ml of PBS/BSA.
5. Centrifuge at 400 g for 5 minutes.
6. Discard supernatant and resuspend pellet in an appropriate amount of
7. PBS/BSA.

(c) Adherent tissue culture cell lines

1. Prepare PBS/BSA buffer (phosphate buffered saline pH 7.4 and 1% BSA).
2. Harvest cells by gentle scraping using 2 ml of PBS/BSA buffer.
3. Transfer cells to a 15 ml conical tube and add buffer up to 10 ml.
4. Centrifuge at 400 g for 5 minutes.
5. Discard supernatant and resuspend pellet in fresh PBS/BSA (10 ml).
6. Centrifuge at 400 g for 5 minutes.
7. Discard supernatant and resuspend pellet in an appropriate amount of
8. PBS/BSA buffer.

(d) Preparing cells from solid/lymphoid tissues

1. Place tissue on a sterile Petri dish. Remove cells by gently perfusing the tissue using a syringe and needle containing approximately 15 ml of PBS/BSA (phosphate buffered saline pH 7.4 and 1% BSA).
2. Transfer the cell suspension from the Petri dish into a 15 ml conical centrifuge tube.
3. Centrifuge at 400 g for 5 minutes.
4. Discard the supernatant and resuspend the pellet in PBS/BSA.
5. Add 10 ml of ammonium chloride lysis buffer.
6. Mix and incubate for 2 minutes. DO NOT EXCEED THIS TIME.
7. Centrifuge at 400 g for 5 minutes.
8. Add 10 ml of PBS/BSA and mix.
9. Centrifuge again at 400 g for 5 minutes.
10. Discard the supernatant and resuspend the pellet to a final volume of 10 ml with PBS/BSA.
11. Count cells using a hemocytometer.
12. Adjust the cell suspension, if necessary.

Red cell lysis protocol

1 ml of 10 mM EDTA 1% Dextran pH7.4, mix 3 drops to 1 ml blood, let the mix sit at 37 degrees C for 45 min (30 might be enough).  Take the yellow supernatant and mix 2000 RPM, 10 min.  Add 1ml ACK lysis buffer to each blood sample. Mix by pipetting gently up and down. Incubate at room temp. for approx. 10 min.  Spin in the microcentrifuge for 3-4 min. @ 3 - 4,000 x g. Aspirate supernatant carefully. Lymphocytes should sit at bottom. Don’t try to get all the platelets.

ACK Lysis Buffer:

0.15 M NH₄Cl (Sigma Cat. No. 09718) 8.3g

10 mM KHCO₃ (Sigma Cat.No. P9144) 1g

0.1 mM EDTA (Sigma Cat.No. E7889)

You can also buy ACK from Invitrogen, Cambrex, etc

Alternative red cell lysis protocol

Red blood cells from murine peripheral blood or a spleen cell suspension can be lysed using BD Biosciences Pharmingen's PharM Lyse™ (Cat. No. 35221E) solution. Add 2.0 ml of 1X Lysing Solution to the spleen cell suspension or per 200µl of murine peripheral blood. Gently vortex immediately after adding the lysing solution. Incubate at room temperature, protected from light, for 15 min. Centrifuge 200 x g for 5 min. Carefully aspirate and dispose of supernatant, without disturbing the pellet. Resuspend pellet in 1X cold wash buffer (PBS/0.1% NaN3/1.0% fetal bovine serum). Centrifuge at 350 x g for 5 min. Finally, resuspend cell pellet to a concentration of 2 x 10⁷ cells/ml (i.e., 10⁶ cells per 50µl).