title: Protocol to cryopreserve and isolate nuclei from adipose tissue without dimethyl sulfoxide.
abstract: Cryopreservation injuries involve nuclear DNA damage. A protocol for cryopreserving and isolating adipocyte nuclei is proposed. Adipose tissue samples were directly analyzed (NoCRYO-0h), or stored at -196°C for 7 days without 10% dimethyl sulfoxide (DMSO) (CRYO-WO-DMSO) or with DMSO (CRYO-W-DMSO). To determine the effect of DMSO on cryopreservation treatment, adipose tissue samples were stored at 4°C for 24 h with 10% DMSO (NoCRYO-W-DMSO-24h) and without (NoCRYO-WO-DMSO-24h). Samples were processed in isolation buffer, and nuclear integrity was measured by flow cytometry. The coefficient of variation, forward scatter, side scatter, and number of nuclei analyzed were evaluated. Pea (Pisum sativum) was used to measure the amount of DNA. All groups contained similar amounts of DNA to previously reported values and a satisfactory number of nuclei were analyzed. CRYO-W-DMSO presented a higher coefficient of variation (3.19 ± 0.09) compared to NoCRYO-0h (1.85 ± 0.09) and CRYO-WO-DMSO (2.02 ± 0.02). The coefficient of variation was increased in NoCRYO-W-DMSO-24h (3.80 ± 0.01) compared to NoCRYO-WO-DMSO-24h (2.46 ± 0.03). These results relate DMSO presence to DNA damage independently of the cryopreservation process. CRYO-W-DMSO showed increased side scatter (93.46 ± 5.03) compared to NoCRYO-0h (41.13 ± 3.19) and CRYO-WO-DMSO (48.01 ± 2.28), indicating that cryopreservation with DMSO caused chromatin condensation and/or nuclear fragmentation. CRYO-W-DMSO and CRYO-WO-DMSO presented lower forward scatter (186.33 ± 9.33 and 196.89 ± 26.86, respectively) compared to NoCRYO-0h (322.80 ± 3.36), indicating that cryopreservation reduced nuclei size. Thus, a simple method for cryopreservation and isolation of adipocyte nuclei causing less damage to DNA integrity was proposed.
authors.affiliation: Departamento de Biologia, Universidade Federal de Juiz de Fora, Juiz de Fora, MG, Brasil.
authors.forename: M M
authors.initials: MM
authors.lastname: Almeida
authors.affiliation: Departamento de Biologia, Universidade Federal de Juiz de Fora, Juiz de Fora, MG, Brasil.
authors.forename: L C J
authors.initials: LC
authors.lastname: Caires
authors.affiliation: Departamento de Biologia, Universidade Federal de Juiz de Fora, Juiz de Fora, MG, Brasil.
authors.forename: C M
authors.initials: CM
authors.lastname: Musso
authors.affiliation: Departamento de Biologia, Universidade Federal de Juiz de Fora, Juiz de Fora, MG, Brasil.
authors.forename: J M S
authors.initials: JM
authors.lastname: Campos
authors.affiliation: Departamento de Biologia, Universidade Federal de Juiz de Fora, Juiz de Fora, MG, Brasil.
authors.forename: C M C
authors.initials: CM
authors.lastname: Maranduba
authors.affiliation: Departamento de Parasitologia, Microbiologia e Imunologia, Universidade Federal de Juiz de Fora, Juiz de Fora, MG, Brasil.
authors.forename: G C
authors.initials: GC
authors.lastname: Macedo
authors.affiliation: Departmento de Física, Universidade Federal de Juiz de Fora, Juiz de Fora, MG, Brasil.
authors.forename: J P R F
authors.initials: JP
authors.lastname: Mendonça
authors.affiliation: Departamento de Biologia, Universidade Federal de Juiz de Fora, Juiz de Fora, MG, Brasil raul.garcia@ufjf.edu.br.
authors.forename: R M G
authors.initials: RM
authors.lastname: Garcia
mesh_terms: D000273:Adipose Tissue / Q000166:cytology / Q000378:metabolism*; D000818:Animals; D002467:Cell Nucleus / Q000235:genetics*; D015925:Cryopreservation / Q000379:methods*; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D004121:Dimethyl Sulfoxide; D005434:Flow Cytometry; D018532:Pisum sativum / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar
chemical_list: D004247:DNA; D004121:Dimethyl Sulfoxide