A Comprehensive Protocol for the Collection, Differentiation, Cryopreservation, and Resuscitation of Primary Murine
Abby M Luu1, Kelly M Shepardson2, Agnieszka Rynda-Apple1
1Department of Microbiology and Cell Biology, Montana State University, Bozeman, Montana, USA.
Background:
The field of immunology has undoubtedly benefited from the in vitro use of cell lines for immunological studies; however, due to the "immortal" nature of many cell lines, they are not always the best model. Thus, direct collection and culture of primary cells from model organisms is a solution that many researchers utilize. To the best of our knowledge, there is not a singular protocol which encompasses the entire process of bone marrow cell collection through cryopreservation and resuscitation of cells from a murine model.
Methods:
Bone marrow cells were collected from mice with a C57BL6 genetic background. Cells were differentiated using L929 conditioned media. Cells were assessed using a combination of microscopy, differential staining, immunocytochemistry, and trypan blue. Results: Primary murine BMDMs that underwent cryopreservation followed by resuscitation retained a high degree of viability. Furthermore, these BMDMs retained on overall ability to clear S. aureus.
Results:
Primary murine BMDMs that underwent cryopreservation followed by resuscitation retained a high degree of viability. Furthermore, these BMDMs retained on overall ability to clear S. aureus.
Conclusion:
Crypopreserved and resuscitated primary murine BMDMs were viable and retained their pverall S. aureus clearance ability.
