Serial Passaging of RAW 264.7 Cells Modulates Intracellular AGE Formation and Downregulates RANKL-Induced In Vitro Osteoclastogenesis

Abstract

The passage number of cells refers to the number of subculturing processes that the cells have undergone. The effect of passage number on morphological and phenotypical characteristics of cells is of great importance. Advanced glycation end products have also been associated with cell functionality and characteristics. Murine monocyte RAW 264.7 cells differentiate into osteoclasts upon receptor activation caused by nuclear factor-kappa-Β ligand (RANKL) treatment. This study aims to identify the role of passage number on intracellular advanced glycation end products (AGEs) formation and osteoclastogenic differentiation of RAW 264.7 cells. Western blotting was performed to check intracellular AGE formation along with fluorometric analysis using a microplate reader. Tartrate-resistant acid phosphatase (TRAP) staining was performed to check osteoclastogenic differentiation, and qPCR was realized to check the responsible mRNA expression. Immunofluorescence was used to check the morphological changes. Intracellular AGE formation was increased with passaging, and the higher passage number inhibited multinucleated osteoclastogenic differentiation. Osteoclastogenic gene expression also showed a reducing trend in higher passages, along with a significant reduction in F-actin ring size and number. Lower passages should be used to avoid the effects of cell subculturing in in vitro osteoclastogenesis study using RAW 264.7 cells.

Exploring the Biological Functions and Anti-Melanogenesis of Phallus indusiatus for Mushroom-Based Cosmetic Applications

“…The magnitude of the anti-inflammatory effect was found to be dependent on the extraction method used. The amount of released NO was dramatically different, which could be explained in terms of cell passaging [46]. These findings shed light on the cosmeceutical properties of P. indusiatus for mushroom-based cosmetic product development.…”

Design, synthesis, antibacterial activity evaluation and molecular docking study of pleuromutilin derivatives bearing amide side chains

“…RAW 264.7 cells are mouse mononuclear macrophage leukemia cells. This cell line is obtained after inducing tumors in BALB/c mice by Abelson murine leukemia virus, and it is also one of the commonly used cells to establish inflammatory cell models (Lucy et al, 2022). Most compounds produced slight inhibitory effect on the viability of RAW 264.7 cells at the concentration of 8.0 μg/ml.…”

Brucella Manipulates Host Cell Ferroptosis to Facilitate Its Intracellular Replication and Egress in RAW264.7 Macrophages

“…Compared to primary macrophages, the RAW264.7 cell line has many advantages: wide availability, easy to operate, homogeneous genetic background, and functional stability [62][63][64]. Therefore, the RAW264.7 cell line is extensively used as a macrophage model for in vitro biology studies [65,66]. Except for that, this cell line also gained popularity as an in vitro model for investigating pathogens' infection, including Brucella [62,63,[67][68][69].…”