Preparing a single cell suspension from a frozen starting sample is critical for optimizing cell isolations by avoiding additional cell loss and enabling the maximum labeling of target cells. Performing cell separation on clumpy cell samples can result in lower recovery and may interfere with proper labeling of the target cells. Samples may sometimes appear "clumpy" when they have been exposed to repeated freeze\/thaw cycles or enzymatic tissue dissociation. These cell clumps occur because environmental stresses can accelerate the rate of cell death within the sample, resulting in the release of "sticky" DNA molecules from the dying cells that can clump neighboring cells together. Adding the endonuclease deoxyribonuclease I (DNase I) into your sample can minimize the presence of free-floating DNA fragments and cell clumps. This protocol describes a method to harvest cells and prepare a clump-free single cell suspension from a frozen cell sample prior to performing cell separation.