Cricetulus griseus
Protocols: Differential Ultracentrifugation Exosome isolation via ultracentrifugation, referred to as UC, was performed modified after Théry et al. 2006. Culture broth was centrifuged 5 min at 500 g and 30 min at 2,000 g thereafter, transferring the respective supernatant. After filtering the supernatant with 0.22 µm minisart filters (Sartorius; Göttingen, Germany), it was concentrated up to 50 X in Vivaspin 20, 100 kDa MWCO centrifugation tubes (Sartorius; Göttingen, Germany). The concentrate was inserted for a two-step ultracentrifugation at 100,000 g for 70 min, washing the exosome-pellet with 1 ml PBS with 1 mM PMSF. The resulting pellet was taken up in pre-filtered PBS with 1 mM PMSF. For AFM application the exosomes were taken up in TE-buffer (10 mM Tris-HCl, 1 mM EDTA) with 1 mM PMSF instead. For the growth phase experiment, microvesicles were sedimented at 20,000 g for 30 min prior to 0.22 µm filtration and washed once. The pellet was taken up in 100 µl PBS with 1 mM PMSF. Host cell protein was concentrated from the 100 kDa MWCO flow through in 3 kDa MWCO centrifugation tubes (Sartorius; Göttingen, Germany) and precipitated twice with 90 % acetone. Lower Speed Centrifugation Cells were sedimented at 300 g for 10 min. Debris and larger microparticles were subsequently depleted centrifuging the supernatant for 30 min at 2,000 g and then for 45 min at 10,000 g. Transferring the supernatant, exosomes were then sedimented for 3 h at 20,000 g, washed in 3 ml PBS with 1 mM PMSF, and again centrifuged at 20,000 g for 3 h. The resulting pellet was resuspended in 100 µL PBS with 1 mM PMSF. ExoQuick-TC™ The ExoQuick-TC™ exosome isolation kit (System Biosciences; Palo Alto CA, USA) was used according to the manufacturer´s instructions on concentrated culture supernatant. Cells and bigger particles were removed by centrifugation at 3,000 g for 15 min. The transferred supernatant was then added with 1/6 volumes of ExoQuick-TC™ precipitation reagent. The reagent was thoroughly mixed with the supernatant and stored at 4 °C overnight. The next day, the sample was centrifuged twice at 1,500 g for 30 min and 5 min, respectively, to take off the supernatant. The pellet was resuspended in 100 µl PBS, supplemented with 1 mM PMSF. PEG precipitation Precipitation with polyethylene glycol (PEG6000) was carried out as noted by M. Rider et al. 2016. Culture broth was centrifuged 5 min at 500 g and 30 min at 2,000 g, carrying over the supernatant. The precipitation mixture containing 16 % PEG with an average molecular weight Mn of 6000 g mol-1 (Merck; Darmstadt, Germany) and 1 mM NaCl, was added 1:1 (v/v) to the sample, thoroughly mixed and stored at 4 °C overnight. The next day, precipitated components were sedimented by centrifugation at 4,000 g for 75 min. After removing the supernatant, the tube was inverted for 10 min and tapped onto cellulose to remove the remaining liquid. The pellet was taken off in filtered, 1 mM PMSF supplemented PBS and transferred into a 13 ml polypropylene tube (Beckman Coulter; Brea CA, USA) before being centrifuged at 100,000 g for 70 min using a TH-641 rotor (Thermo-Fisher Scientific; Waltham MA, USA) in an Ultra-Pro 80 centrifuge (Thermo-Fisher Scientific; Waltham MA, USA). The resulting exosome-pellet was resuspended in particle-free PBS with 1 mM PMSF. Sodium Acetate Precipitation The used workflow complies with the protocol reported by Brownlee et al. 2014. Centrifugation of the supernatant took place at 500 g for 30 min and another 30 min at 10,000 g, transferring the supernatant. 0.1 volumes of 1 M NaOAc solution (pH 4.75) were added to the cleared supernatant and the mixture was thoroughly shaken and stored 60 min on ice, followed by 5 min at 37 °C. The precipitate was then centrifuged at 5,000 g for 10 min to pellet exosomes, resuspended in 0.1 M NaOAc and again centrifuged for 10 min at 5,000 g to wash them. The resulting pellet was taken off in filtered PBS with 1 mM PMSF. Preparing Exosome and Cell Lysates Cell samples for lysis and protein extraction were collected parallelly to exosome isolation by taking off a volume that fits 107 cells and washing the cells twice for 5 min at 200 g in cold PBS. The cell and EV pellets were stored at -80 °C until thawing. Chinese hamster ovary (CHO) K1 cells (strain ATCC 61-CCL) were cultivated in suspension in Erlenmeyer Shake Flasks at 185 rpm, 37 °C, 5 % CO2 and 80 % humidity in a Mytron cell culture incubator (Memmert; Schwabach, Germany). To determine viable cell density (VCD) and viability, they were counted with a Cedex™ automated cell counter device (Roche; Basel, Switzerland). Cultivation took place in serum-free TCX6D medium (Xell; Bielefeld, Germany), supplemented with 8 mM glutamine. Culture Broth Harvest If not delivered sterile and particle-free as part of a commercial kit, respective buffers and solutions were filtered using filter papers (Sartorius; Göttingen, Germany). If not otherwise stated, all steps took place on ice or at 4 °C. Cell cultures were harvested for exosome separation during the late exponential growth phase to ensure a high exosome density without possibly interfering starvation effects. For total RNA extraction cell pellets were resuspended in 600 µl TRI Reagent (Zymo Research) and EV pellets were resuspended in 100 µl PBS and 300 µl TRI Reagent was added. The lysed samples were mixed with 200 µl (EV 100 µl) chloroform and were centrifuged at 12,000 g, 4 °C for 15 min. The aqueous phase was taken of and extracted again with 500 µl (EV 300 µl) TRI Reagent and 200 µl (EV 100 µl) chloroform. RNA was precipitated from the aqueous phase by incubation with the same volume isopropanol and centrifuged at 12,000 g, 4 °C for 10 min. The RNA pellet was washed with 1 mL (EV 300 µl) 75 % ethanol twice. After drying, the pellet was resuspended in 50 µl (EV 10 µl) RNase-free water. 100 ng exosomal RNA were used to prepare Lexogen small strand-specific RNA-Seq libraries for Illuminawith the Small RNA-Seq Library Prep Kit for Illumina (Lexogen) according to the manufacturer's instructions (Lexogen catalogue number 052.24). Size selection of small RNA-Seq libraries and adapter removal was performed with BluePippin (Sage Science) using a 3% agarose standard DNA size selection cassette selecting fragments ranging from 137 to 162 nt, which includes inserts from 15 to 40 nt. Prior to sequencing, the indexed libraries were quantified using the the Qubit (ThermoFisher Scientific) and the Agilent Bioanalyzer 2100 (Agilent).
ENA first public
2021-01-18
ENA last update
2020-12-10
External Id
SAMEA7702581
INSDC center alias
Center for Biotechnology (CeBiTec), Bielefeld University
INSDC center name
Center for Biotechnology (CeBiTec), Bielefeld University
INSDC first public
2021-01-18T04:08:54Z
INSDC last update
2020-12-10T16:49:57Z
INSDC status
public
Submitter Id
E-MTAB-9894:Exo_Exp_1
broker name
ArrayExpress
cell line
CHO-K1 (strain ATCC 61-CCL)
common name
Chinese hamster
fraction
exosomes (EXO)
genotype
wild type genotype
growth condition
logarithmic phase (log)
organism part
ovary
replicate
1
sample name
E-MTAB-9894:Exo_Exp_1