Buffer Preparation

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This section describes buffer preparation for an input of 1 million cells in a 200 μl volume. Optimize the volume based on specific cell numbers, sample numbers, or Cell Suspension Buffer (CSB) volume.

• Absolute methanol (MeOH)
• DMSO
• DSP
• 1.5 ml low retention microcentrifuge tube
• 1000 μl pipette tips

Fixative Preparation (Prepare Fresh)

1. Prepare the DSP solution as follows.
a. Dissolve DSP into DMSO at 50 mg/ml concentration.

For example, dissolve 1 mg of DSP in 20 μl 100% Anhydrous DMSO to generate 20 μl of DSP solution.

One 20 μl aliquot of DSP solution can fix up to 10^6 cells.

b. Vortex to mix, and then centrifuge briefly.
2. In a 1.5 ml tube, combine the following volumes to prepare a working solution of fixative.
• DSP solution (20 μl)
• Chilled 100% MeOH (800 μl)
3. Using a 1000 μl pipette, gently pipette to mix.
4. Keep the fixative on ice until use.