Shipping and Storage

For short-term storage (< 1 month), store in -80°C freezer.  For long-term storage (> 1 month), store in vapor phase of liquid nitrogen storage tank.

WARNING: Please store vials in vapor phase of liquid nitrogen storage tank. Vials may explode if stored in liquid phase of liquid nitrogen storage tank.

Reagent Requirement: 1× PBS and 0.4% Trypan Blue.

Thawing Cells

1. Clean the frozen vial with 70% alcohol before thawing. In a biosafety hood, twist the cap a quarter-turn to relieve pressure, and then retighten the cap.
2. In a 37°C water bath, quickly thaw the vial. Be careful not to submerge the entire vial in the water bath. Do not remove the vial until a tiny ice-crystal is left. Clean the outside of the vial with 70% alcohol.
3. In a biosafety hood, measure the cell suspension volume (V) or refer to Table 1.

Table 1: Typical Cell Suspension Volumes

1. Prepare the solution according to Table 2. For instance, pipette 10µL of sample and mix with 10µL of 0.4% Trypan Blue and 20µL of 1× PBS for a unit size of 5×106.
2. Load 10µL of the mixed solution onto a hemacyto-meter and count the number of cells.

Table 2: Recommended Sample Dilution

Counting Cells

N = # of cells counted on all 4 squares of a hemacyto-meter

D = dilution factor

V= cell suspension volume

Equation for Cell Count:

# of live cells/ vial = N / 4 x D × V (mL) = _____ × 104

Equation for Viability:

# of live cells/ (Live cells + Dead cells) × 100% = ______%

**This is a very important step to determine if the percentage of viable cells matches what C&M LABPRO minimum claims.

Alternative Method

This method aims to reduce cell clumping prior to cell count.

Note: Each cell-wash cycle reduces cell count & viability.

General Medium Requirements

1. IMDM, MEM, or RPMI-1640 containing 10% FBS
2. DNase I (StemCell Technology, Cat. No. 07900;1mg/ml in PBS)

Procedure

1. Add DNase I to a 50mL conical tube prior to transferring cells.

• To thaw purified cells for culture purpose only, a total of 100µL DNase solution is needed.
• To thaw mononuclear cells for cell culture purpose only, a total of 300µL DNase solution is needed.

1. Thaw the vial in a 37°C water bath. Transfer thawed cells into the 50mL conical tube, which contains DNase I (step 1).

1. Rinse the vial with 1 mL culture medium containing 10% FBS. In the 50ml conical tube, slowly add the rinse drop by drop (5 seconds per drop) to the cells, while gently shaking the tube.

1. Next, slowly add the medium drop by drop (5 seconds per drop) to the cells until the total volume is 15-20 ml. Gently invert to mix.
2. Centrifuge the cell suspension at 200g at room temperature for 15 minutes.
3. Using a pipette, carefully remove most of the supernatant (save the supernatant in a second tube). Leave a few milliliters of the supernatant behind so the cell pellet is not disturbed. Gently re-suspend the cell pellet in the remaining few milliliters of medium.
4. While gently shaking the tube, slowly add an additional 15-20ml of fresh medium to the tube.
5. Centrifuge the cell suspension at 200g at room temperature for 15 minutes.
6. Using a pipette, carefully remove all but 2ml of the supernatant. Gently re-suspend the cell pellet in the remaining 2 mL of medium and count. If the cell count is lower than expected, centrifuge the supernatant saved in Step 9 at a higher speed, count and combine if necessary. The cells are ready for use in your experiment.