Long-term cell storage of RAW 264.7 cells in a deep freezer dampens M2 marker expression

RAW 264.7, a well-established cell line, is widely used in research; however, it is easily polarized upon subtle changes in environmental conditions or repeated passages. As the effects of cell storage conditions and duration on their phenotypic changes remain unknown, we investigated the effects of long-term storage at -80 °C on M2 marker expression in RAW 264.7 cells after stimulation with IL-4. Cells, stored in a - 80 °C deep freezer for a few years (defined as "long-stored cells") and newly purchased cells (referred to as "new cells"), were compared through flow cytometry for CD68-positive naïve macrophages (M0) and the expression of three types of M2 markers, CD206-FITC, CD206-PE, and Arginase-1-Alexa Fluor 488, in the M2 state. CD68 positivity was similar between the M0 macrophage types. After stimulation with IL-4, the proportion of CD206-FITC-positive cells was 5.0 ± 0.7 % and 61.5 ± 3.1 % in long-stored cells and new cells, respectively (p < 0.0001), while that of CD206-PE-positive cells was 9.2 ± 0.2 % and 69.8 ± 0.6 %. Similarly, the proportion of Arginase-1- Alexa Fluor 488-positive cells was 3.0 ± 0.1 and 9.0 ± 9.6 %, respectively (p = 0.0006). Overall, these results demonstrate that long-term storage of RAW 264.7 in a deep freezer affects their M2 marker expression. These results highlight the need to consider the effect of cell storage conditions and duration on experimental results.