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Amicon ultra pl 3

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The Amicon Ultra-PL 3 is a centrifugal filter unit designed for the rapid concentration and desalting of biological samples. It features a regenerated cellulose membrane with a molecular weight cut-off of 3 kDa.

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Lab products found in correlation

100 mm cell dishes, by Corning (5 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions) Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions) Countess, by Thermo Fisher Scientific (1 mentions) Mic 101, by Embrient Inc (1 mentions) Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions) Muse count viability assay kit, by Merck Group (1 mentions) Pgc 1α, by OriGene (1 mentions) Dmem low glucose medium, by Thermo Fisher Scientific (1 mentions) Nanodrop nd 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)

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Amicon Ultra

12 protocols using amicon ultra pl 3

1

Evaluating the Impact of miR-122 on ASCs

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ASCs were transfected with miR-122 (Exiqon, Germatown, MD) per well mixed with the Lipofectamine RNAiMAX Reagent (Thermo). After 72hr of transfection, the cells were morphologically observed by the inverted microscope. The cell numbers of the experimental groups were counted automatic cell counter (Countess®, Invitrogen, San Diego, CA, United States) using trypan blue solution. Transfected cells were processed for cell phenotyping or differentiated into three-lineage induction.
ASCs with or without miR-122 transfection were grown in a 100 mm cell dishes (Corning Glass Works, Corning, NY, United States). After reaching 70%-80% confluence, 1.0 × 106 ASCs were cultured in 5 mL serum-free low-glucose DMEM for 48 h. Therefore, to obtain 0.2 mL amount of secretome from 1.0 × 106 ASCs, the conditioned media were concentrated 25-fold using ultra filtration units with a 3-kDa molecular weight cutoff (Amicon Ultra-PL 3; Millipore, Bedford, MA, United States). We then injected 0.1 mL amount of secretome per mouse. This means that one mouse is injected with the secretome obtained from 5 × 105 ASCs. In this study, NCM refers to the secretome shed from ASCs after 48 h of incubation, and MCM refers to the secretome shed from miR-122-transfected ASCs after 48 h of incubation.
Kim K.H., Lee J.I., Kim O.H., Hong H.E., Kwak B.J., Choi H.J., Ahn J., Lee T.Y., Lee S.C, & Kim S.J. (2019). Ameliorating liver fibrosis in an animal model using the secretome released from miR-122-transfected adipose-derived stem cells. World Journal of Stem Cells, 11(11), 990-1004.
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2

Conditioned Medium Production from Stem Cells

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CM was prepared according to our previous reports [14 (link)]. After the Ctrl-hMSCs or ISL1-hMSCs had reached greater than 80% confluence, the cells were cultured with serum-free DMEM for another 24 h. The medium from 106 cells yielded 6 ml of primary CM, which was further de-salted and concentrated 30-fold by centrifugation (4000 × g for 30 min at 4 °C) using ultrafiltration units with a 3-kDa molecular weight cutoff (Amicon Ultra-PL 3; Millipore, Billerica, MA, USA), yielding 200 μl of concentrated CM. Serum-free DMEM (de-salted and concentrated 30-fold) served as a vehicle control. CM was used immediately or stored at −80 °C.
Xiang Q., Liao Y., Chao H., Huang W., Liu J., Chen H., Hong D., Zou Z., Xiang A.P, & Li W. (2018). ISL1 overexpression enhances the survival of transplanted human mesenchymal stem cells in a murine myocardial infarction model. Stem Cell Research & Therapy, 9, 51.
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3

Hypoxic Preconditioning of ASCs

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ASCs were re-fed with serum-free low-glucose DMEM. ASCs were then cultured under either normoxic (21% pO2) or hypoxic (10%, 5%, and 1% pO2) conditions. The duration of HP was determined to be 24 h, during which the expression of signaling intermediates was highly amplified (Additional file 1: Figure S1). Hypoxic preconditioned conditioned media (CMs) were obtained by placing the ASCs in a hypoxic chamber (MIC-101; Billups-Rothenberg Inc., San Diego, CA, USA) at 37 °C for 24 h. Each CM was then concentrated 25-fold, using ultrafiltration units (Amicon Ultra-PL 3; Millipore, Bedford, MA, USA) with a 3-kDa cutoff, and the concentrated CM was considered the secretome. The secretomes with normoxic or various HP were stored at −80 °C until use.
Lee S.C., Kim K.H., Kim O.H., Lee S.K., Hong H.E., Won S.S., Jeon S.J., Choi B.J., Jeong W, & Kim S.J. (2017). Determination of optimized oxygen partial pressure to maximize the liver regenerative potential of the secretome obtained from adipose-derived stem cells. Stem Cell Research & Therapy, 8, 181.
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4

ASC Secretome Concentration Protocol

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ASCs were cultured in 100 mm cell dishes (Corning Glass Works, Corning, NY). Upon reaching 70%–80% confluence, 1.0 × 106 ASCs were incubated in 7 mL of serum-free low-glucose DMEM for 24 h. To obtain a 0.2 mL volume of secretome from 1.0 × 106 ASCs, the conditioned media were concentrated 25-fold using ultrafiltration units with a 3-kDa molecular weight cutoff (Amicon Ultra-PL 3; Millipore, Bedford, MA).
Kim S.J., Kim O.H., Hong H.E., Ju J.H, & Lee D.S. (2024). Etanercept-synthesizing adipose-derived stem cell secretome: A promising therapeutic option for inflammatory bowel disease. World Journal of Gastrointestinal Surgery, 16(3), 882-892.
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5

Secretome Preparation and Dosing

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ASCs were grown in a 100 mm cell dishes (Corning Glass Works, Corning, NY, USA). After reaching 70–80% confluence, 1.0 × 106 ASCs were cultured in 5 mL serum-free low-glucose DMEM for 48 h. Therefore, to obtain 0.2mL amount of secretome from 1.0 × 106 ASCs, the conditioned media were concentrated 25-fold using ultra filtration units with a 3-kDa molecular weight cutoff (Amicon Ultra-PL 3; Millipore, Bedford, MA, USA). We then injected 0.1 mL amount of secretome per mouse. This means that one mouse is injected with the secretome obtained from 5 × 105 ASCs. In this study, control secretome refers to the secretome obtained from empty vector-transfected ASCs, and etanercept-secretome refers to the secretome obtained from etanercept-synthesizing ASCs.
Han J.H., Kim O.H., Lee S.C., Kim K.H., Park J.H., Lee J.I., Lee K.H., Hong H.E., Seo H., Choi H.J., Ju J.H, & Kim S.J. (2019). A Novel Hepatic Anti-Fibrotic Strategy Utilizing the Secretome Released from Etanercept-Synthesizing Adipose-Derived Stem Cells. International Journal of Molecular Sciences, 20(24), 6302.
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6

Conditioned Medium from Neural Crest Stem Cells

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To prepare the CM, the NCSCs (5 × 106) were seeded in a 60-mm culture dish. The NCSCs were incubated with serum-free Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich) for 24 h to produce CM. The CM was collected and filtered through a 0.2-μm filter to remove cellular debris. The adherent cells were trypsinized, stained with trypan blue, and counted. The medium obtained from 5 × 106 cells yielded 15 mL of primary CM, which was further desalted and concentrated approximately 25-fold, using ultrafiltration units with a 3-kDa molecular weight cutoff (Amicon Ultra-PL 3; Millipore, Billerica, MA, USA), yielding a final volume of 600 μL CM [9 (link)]. Serum-free DMEM, desalted and concentrated 25-fold, served as the vehicle control [9 (link)].
Peng C.K., Wu S.Y., Tang S.E., Li M.H., Lin S.S., Chu S.J, & Huang K.L. (2017). Protective Effects of Neural Crest-Derived Stem Cell-Conditioned Media against Ischemia-Reperfusion-Induced Lung Injury in Rats. Inflammation, 40(5), 1532-1542.
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7

Preparation of Conditioned Medium from MSCs

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For the preparation of the conditioned medium (CM), MSCs (passage 3–7) were cultured in the presence of their expansion medium until 80% of confluence. Conditioned medium was obtained from supernatants of 8.95 ± 0.46 × 103/cm2 MSCs maintained in RPMI medium supplemented with 0.1% BSA for 16 h. The viability of MSCs after starvation was about 86 ± 0.5% as detected by the Muse® Count &Viability Assay Kit (CTRL normal medium 88,9% of vitality) (Millipore, MA, USA). Supernatant was first centrifuged at 1500 g for 20 min, to remove debris and apoptotic bodies and then concentrated at 4 °C, approximately 200-fold, using ultrafiltration units (Amicon Ultra-PL 3, Millipore) with a 3 kDa molecular weight cut-off as previous described [21 (link)]. After the concentration CM-containing EVs in 1% dimethyl sulfoxide was kept at −80 °C until use.
Collino F., Pomatto M., Bruno S., Lindoso R.S., Tapparo M., Sicheng W., Quesenberry P, & Camussi G. (2017). Exosome and Microvesicle-Enriched Fractions Isolated from Mesenchymal Stem Cells by Gradient Separation Showed Different Molecular Signatures and Functions on Renal Tubular Epithelial Cells. Stem Cell Reviews, 13(2), 226-243.
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8

Adipose-Derived Stem Cell Secretome Preparation

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ASCs were grown in 100-mm cell dishes (Corning Glass Works, Corning, NY, USA). After reaching 70% to 80% confluence, 5.0 × 105 ASCs were cultured in 5 mL of serum-free low-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Scientific, Hemel Hempstead, UK) with or without LPS in low concentration (0.5 ng/mL; Sigma-Aldrich, St. Louis, MO, USA) for 24 hours. In a variety of experiments including intravenous administration of MSCs, 0.1 mL of 1 × 105 to 1.0 × 106 MSCs has been used for an injection into a mouse [17 (link),30 (link)-32 (link)]. In our protocol, the secretome from 5.0 × 105 ASCs was cultured in 5 mL of DMEM. Therefore, to obtain the equivalent amount (0.1 mL) of secretome, the conditioned media were concentrated 25-fold by using ultrafiltration units with a 3-kDa-molecular-weight cutoff (Amicon Ultra-PL 3; Millipore, Bedford, MA, USA) after stimulation with or without LPS. From here on, LPS-CM and CM refer to the 25-fold concentrated conditioned media which had been obtained from ASCs after stimulation with or without LPS for 24 hours.
Lee S.C., Jeong H.J., Lee S.K, & Kim S.J. (2015). Lipopolysaccharide preconditioning of adipose-derived stem cells improves liver-regenerating activity of the secretome. Stem Cell Research & Therapy, 6(1), 75.
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9

Secretome from PGC-1α-Overexpressing ASCs

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ASCs were grown in 100 mm cell dishes (Corning Glass Works, Corning, NY, USA). After reaching 70–80% confluence, the ASCs were transiently transfected with 1 μg pcDNA-PGC-1α. The PGC-1α plasmid was purchased from OriGene Technologies (Rockville, MD, USA). After 24 h, 1.0 × 106 ASCs were cultured in 5 mL serum-free low-glucose DMEM for 24 h. Therefore, to obtain 0.2 mL of secretome from 1.0 × 106 ASCs, the conditioned media were concentrated 25-fold using ultra filtration units with a 3-kDa molecular weight cutoff (Amicon Ultra-PL 3; Millipore, Bedford, MA, USA). We then injected 0.1 mL of secretome per mouse. This means that one mouse was injected with the secretome obtained from 5 × 105 ASCs. In this study, normal secretome refers to the secretome obtained from empty vector-transfected ASCs, and PGC-secretome refers to the secretome obtained from pcDNA-PGC-1 α transfected ASCs.
Lee J., Kim O.H., Lee S.C., Kim K.H., Shin J.S., Hong H.E., Choi H.J, & Kim S.J. (2019). Enhanced Therapeutic Potential of the Secretome Released from Adipose-Derived Stem Cells by PGC-1α-Driven Upregulation of Mitochondrial Proliferation. International Journal of Molecular Sciences, 20(22), 5589.
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10

Hypoxic Conditioning of Adipose-Derived Stem Cells

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ASCs that reached 70%–80% confluence were re-fed with serum free DMEM low glucose medium (Thermo Scientific, Hemel Hempstead, UK) at 37℃ under 5% CO2. HCM was achieved by placing the ASCs in a hypoxic chamber (MIC-101, Billups-Rothenberg Inc., San Diego, CA, USA) with a mixture of 1% O2, 5% CO2, and balanced N2 at 37℃ for 24 hours. Each NCM was then concentrated by 25 fold, using ultrafiltration units (Amicon Ultra-PL 3, Millipore, Bedford, MA, USA) with a 3-kDa cutoff. Concentrated NCM, obtained under either normoxic or hypoxic culturing conditions, are herein termed NCM and HCM, respectively. The NCM and HCM were stored at −80℃ until use.
Hong H.E., Kim O.H., Kwak B.J., Choi H.J., im K.H., Ahn J, & Kim S.J. (2019). Antioxidant action of hypoxic conditioned media from adipose-derived stem cells in the hepatic injury of expressing higher reactive oxygen species. Annals of Surgical Treatment and Research, 97(4), 159-167.
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