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Knockout serum replacement (ksr)

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Knockout Serum Replacement is a cell culture medium supplement used in research applications. It is designed to support the growth and maintenance of embryonic stem cells and other cell lines that require serum-free conditions. The product provides essential nutrients and growth factors to cells without the inclusion of animal-derived serum components.

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Lab products found in correlation

Knockout serum replacement (ksr), by Thermo Fisher Scientific (4 mentions) β mercaptoethanol, by Merck Group (4 mentions) Fibroblast growth factor 2 (fgf2), by Thermo Fisher Scientific (3 mentions) Pen strep, by Merck Group (2 mentions) L proline, by Merck Group (2 mentions) Probumin, by Merck Group (2 mentions) Beta meracptoethnaol, by Merck Group (2 mentions) 13c615n4 arg10, by Merck Group (2 mentions) 13c615n2 lys8, by Merck Group (2 mentions) Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions) Bone morphogenetic protein 4 (bmp4), by Thermo Fisher Scientific (2 mentions) β mercaptoethanol, by Thermo Fisher Scientific (2 mentions) Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (2 mentions) L glutamine, by Merck Group (2 mentions) Bfgf2, by Thermo Fisher Scientific (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Vascular endothelial growth factor (vegf), by Thermo Fisher Scientific (1 mentions) Penicillin streptomyocin, by Merck Group (1 mentions) Human fgf, by Merck Group (1 mentions) Hydrogel, by Corning (1 mentions)

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50x serum replacement (2 citations) Knockout serum (2 citations)

13 protocols using knockout serum replacement (ksr)

1

iPSC Culturing and Protein Harvesting

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The iPSC line used in this study (CTL1R-1) was derived and initially cultured as described in Adamo et al. [45 (link)]. Cells were growing in single-cell condition in mTeSR-1 and then adapted for 2 passages in a custom medium, composed as follows: DMEM, Knockout Serum Replacement 15 % (Sigma), Pen-Strep 1 %, Non-essential aminoacids 1 %, Glutamine 1 %, Probumin 0.5 % (Millipore), beta-meracptoethnaol 0.1 mM, L-Proline 500 mg/l (Sigma), FGF2 10 ng/ml (Peprotech). The medium was conditioned for 24 hours on a mouse embryonic fibroblast layer inactivated with mitomycin-C and filtered before use. In the SILAC version of the medium a custom DMEM (Lonza) without arginine and lysine was complemented with 84 mg/l 13C615N4 Arg10 (Sigma) and 146 mg/l 13C615N2 Lys8 (Sigma). Cells were scraped and washed in cold PBS upon reaching 70 % confluence approximately for protein harvest.
Cells were counted using a Bürker chamber with Trypan blue counting 5 fields and averaging. Each count was done in duplicate.
Röst H.L., Liu Y., D’Agostino G., Zanella M., Navarro P., Rosenberger G., Collins B.C., Gillet L., Testa G., Malmström L, & Aebersold R. (2016). TRIC: an automated alignment strategy for reproducible protein quantification in targeted proteomics. Nature methods, 13(9), 777-783.
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2

Vascular Differentiation of Mouse ESCs

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For vascular differentiation, mESC were seeded at a seeding density of 2 × 105 cells/cm2 and cultured with differentiation media for seven days. Differentiation media was made up of α-MEM with L-glutamine (Life Technologies), 20% knockout serum replacement (Sigma), 100μg/mL penicillin/Streptomyocin (Sigma), 10mM non-essential amino acids (Life Technologies) and 50uM β-mercaptoethanol (Life Technologies). Growth factors were added to the basal media to induce a vascular differentiation using different combinations of VEGF (Peprotech, 30ng/mL), bFGF2 (Peprotech, 12.5ng/mL), GSK beta inhibitor chir99021 (Tocris Bioscience 4423, 3μM), or BMP4 (Peprotech, 12ng/mL). VEGF, bFGF2 and chir99021 was used as the standard media condition, unless otherwise stated. Medium was exchanged 50% every day over seven days.
Dorsey T.B., Kim D., Grath A., James D, & Dai G. (2018). Multivalent Biomaterial Platform to Control the Distinct Arterial Venous Differentiation of Pluripotent Stem Cells. Biomaterials, 185, 1-12.
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3

Multicellular Tumor Spheroids for Pancreatic Cancer

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Multicellular tumor spheroids from PANC1, KLM-1, L3.6pl, MiaPaCa-2, and the primary low-passage tissue culture line SB.06 derived from the primary tumor of a pancreatic cancer patient were created as previously described.29 (link) In brief, 30,000 to 100,000 cells grown under conventional monolayer 2D conditions were seeded in 15-20 mL stem cell media (SCM) onto nonadherent T75 flasks coated with hydrogel (Corning Life Sciences, Chelmsford, Mass). 500mL stem cell media (SCM) contained 1:1 DMEM: F12 media (cat. #11320-033, Life Technologies, Carlsbad, CA), 5mL 100x ITS media supplement (cat. #13146, Sigma), 2g bovine serum albumin (0.40%) (cat. #A2153, Sigma), 1% KnockOut™ Serum Replacement (cat. #10828028), 20 ng/mL human EGF (cat. #E9644, Sigma), and 10ng/mL human FGF (cat. #F0291, Sigma). Spheroids were grown at 37°C for 14 days. Spheroids were harvested and mechanically dissociated via gentle up-and-down pipetting for 2-3 minutes using a motorized Levo Plus® Pipette Filler (cat. #74020002; Scilogex, Rocky Hill, CT) set on low speed. Single cell suspensions were confirmed under the microscope and counted prior to re-seeding in SCM. Spheres were allowed to re-form for 48-72 hrs prior to drug testing or transfection with siRNA (abbreviated spheroid protocol).29 (link),30 (link)
Bian Y., Teper Y., Griner L.A., Aiken T.J., Shukla V., Guha R., Shinn P., Xin H.W., Pflicke H., Powers A.S., Li D., Jiang J.K., Patel P., Rogers S.A., Aubé J., Ferrer M., Thomas C.J, & Rudloff U. (2019). Target deconvolution of a multikinase inhibitor with anti-metastatic properties identifies TAOK3 as a key contributor to a cancer stem cell-like phenotype. Molecular cancer therapeutics, 18(11), 2097-2110.
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4

iPSC Culturing and Protein Harvesting

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The iPSC line used in this study (CTL1R-1) was derived and initially cultured as described in Adamo et al. [45 (link)]. Cells were growing in single-cell condition in mTeSR-1 and then adapted for 2 passages in a custom medium, composed as follows: DMEM, Knockout Serum Replacement 15 % (Sigma), Pen-Strep 1 %, Non-essential aminoacids 1 %, Glutamine 1 %, Probumin 0.5 % (Millipore), beta-meracptoethnaol 0.1 mM, L-Proline 500 mg/l (Sigma), FGF2 10 ng/ml (Peprotech). The medium was conditioned for 24 hours on a mouse embryonic fibroblast layer inactivated with mitomycin-C and filtered before use. In the SILAC version of the medium a custom DMEM (Lonza) without arginine and lysine was complemented with 84 mg/l 13C615N4 Arg10 (Sigma) and 146 mg/l 13C615N2 Lys8 (Sigma). Cells were scraped and washed in cold PBS upon reaching 70 % confluence approximately for protein harvest.
Cells were counted using a Bürker chamber with Trypan blue counting 5 fields and averaging. Each count was done in duplicate.
Röst H.L., Liu Y., D’Agostino G., Zanella M., Navarro P., Rosenberger G., Collins B.C., Gillet L., Testa G., Malmström L, & Aebersold R. (2016). TRIC: an automated alignment strategy for reproducible protein quantification in targeted proteomics. Nature methods, 13(9), 777-783.
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5

Stepwise Differentiation of Mesenchymal Stem Cells

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First, 4×104 cells were seeded in six-well plates with HDF medium. After 48 h, the standard medium was removed, the cells were washed with PBS, and the Stage 1 medium was added: DMEM/F12, 2.5 mM L-glutamine, 10% fetal bovine serum, 1% non-amino acid essential, 10% Knockout Serum Replacement (Gibco), 100 U/mL of penicillin, 100 μg/mL of streptomycin, 1 mM VPA, 1 μM 5AZ, 5 μM CHIR99021, a GSK-3α inhibitor (Sigma-Aldrich), and 50 μg/mL ascorbic acid (Sigma-Aldrich) with change every 48 h. At day 10 of culture, cells were harvested and re-plated in a six-well plate. After 15 days of culture in Stage 1 medium, the medium was switched to Stage 2 medium: DMEM/F12, 2.5 mM L-glutamine, 10% fetal bovine serum, 1% non-essential amino acids, 10% Knockout Serum Replacement, 100 U/mL of penicillin, 100 μg/mL of streptomycin, 1 mM VPA, 1 μM 5AZ, 5 μM CHIR99021, 50 μg/mL ascorbic acid, 0.5 μM A83-01 (Sigma-Aldrich), and 25 ng/mL of bFGF (Invitrogen) with medium change every 48 h for additional 15 days.
Aguirre-Vázquez A., Salazar-Olivo L.A., Flores-Ponce X., Arriaga-Guerrero A.L., Garza-Rodríguez D., Camacho-Moll M.E., Velasco I., Castorena-Torres F., Dadheech N, & Bermúdez de León M. (2021). 5-Aza-2′-Deoxycytidine and Valproic Acid in Combination with CHIR99021 and A83-01 Induce Pluripotency Genes Expression in Human Adult Somatic Cells. Molecules, 26(7), 1909.
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6

Muscle-Derived Secretome for Neuronal Support

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Once myotubes were matured and 10~20% of myotubes began to twitch autonomously, approximately 4 days in muscle differentiation media (see above), the media was switched back to pre-muscle-conditioned media. The pre-muscle-conditioned media with the contracting myotubes was collected using 0.22 μm filters every 24 hours for 8 days and stored at −80 °C. For the control, pre-muscle-conditioned media was collected from a culture dish without muscle cells, treated, incubated, and stored the same way as with muscle cells. The final forms of RM and CM consisted of these media combined with Neurobasal medium at a volume ratio of 1:1, 10% KnockOut serum replacement, 1% GlutaMAX (all from Gibco), and 1% Penicillin-Streptomycin with ice-cold 0.1 mM β-mercaptoethanol (Gibco), 10 ng/ml glial-derived neurotrophic factor (Neuromics), and 10 ng/ml ciliary neurotrophic factor (Sigma).
For the collection of Ast-RM and Ast-CM, primary hippocampal neurons and astrocytes were cultured in basal media consisting of Advanced DMEM/F-12 and Neurobasal medium at a volume ratio of 1:1 10% KnockOut serum replacement, 1% GlutaMAX, and 1% Penicillin-Streptomycin. Once the confluency reached 100%, the media was replenished with RM or CM for the collection of Ast-RM or Ast-CM, respectively. The media was collected using 0.22 μm filters every 24 hours and stored at −80 °C.
Kaplan D., Ferrari I., Bergami P.L., Mahler E., Levitus G., Chiale P., Hoebeke J., Van Regenmortel M.H, & Levin M.J. (1997). Antibodies to ribosomal P proteins of Trypanosoma cruzi in Chagas disease possess functional autoreactivity with heart tissue and differ from anti-P autoantibodies in lupus. Proceedings of the National Academy of Sciences of the United States of America, 94(19), 10301-10306.
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7

Differentiation of Human Pluripotent Stem Cells

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All media components were from Life Technologies unless otherwise noted. For hPSC culture on mouse embryonic fibroblast (MEF) feeders, the following media were used: MEF (1X high glucose DMEM, 10% fetal bovine serum, 1% (v/v) L-glutamine penicillin/streptomycin). H9/HES3/RiPSC hPSCs (1X DMEM-F12, 20% (v/v) Knockout Serum Replacement, 1% (v/v) non-essential amino acids, 0.5% (v/v) glutamine, 120 μM 2-mercaptoethanol [Sigma]). HSF4 (1X high glucose DMEM+L-Glutamine, 20% (v/v) Knockout Serum Replacement, 1% (v/v) non-essential amino acids, 100 μM 2-mercaptoethanol). All hPSC lines were maintained on feeder layers of mitotically inactivated MEFs (Millipore). All hPSC cultures were supplemented with 30 ng/ml FGF2 (Life Technologies). For culture of hPSCs in the absence of feeders, hPSCs were grown on Matrigel (BD Biosciences) or Geltrex (Life Technologies) in the presence of MEF-conditioned media (MEF-CM; produced by culturing hPSC medium on MEFs for 24 hr followed by sterile filtering), mTeSR2 (Stem Cell Technologies), or Essential 8 (Life Technologies). Cells were routinely passaged every 4–5 days with Accutase and 5 μM Rho kinase inhibitor (Y-27632) (Stemgent) to aid in cell survival. HPSCs were differentiated to early endoderm (EN), mesoderm (ME), and ectoderm (EC) cell populations as previously described (29 ).
Varun D., Srinivasan G.R., Tsai Y.H., Kim H.J., Cutts J., Petty F., Merkley R., Stephanopoulos N., Dolezalova D., Marsala M, & Brafman D.A. (2016). A robust vitronectin-derived peptide for the scalable long-term expansion and neuronal differentiation of human pluripotent stem cell (hPSC)-derived neural progenitor cells (hNPCs). Acta biomaterialia, 48, 120-130.
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8

Culturing hiPSCs and hESC Lines

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hiPSCs and the hESC lines WIBR3 (Whitehead Institute Center for Human Stem Cell Research, Cambridge, MA) and BG01 (NIH code: BG01; BresaGen, Inc., Athens, GA) were maintained on mitomycin C-inactivated mouse embryonic fibroblast feeder layers in hESC medium (DMEM/F12 supplemented with 15% FBS (Hyclone), 5% KnockOut Serum Replacement, 1 mM glutamine, 1% nonessential amino acids, 0.1 mM β-mercaptoethanol (Sigma) and 4 ng ml−1 FGF2 (R&D systems))30 (link). Cultures were passaged every 5-7 days either by trituration or enzymatically with collagenase type IV (Invitrogen; 1.5 mg ml−1).
Dettmer U., Newman A.J., Soldner F., Luth E.S., Kim N.C., von Saucken V.E., Sanderson J.B., Jaenisch R., Bartels T, & Selkoe D. (2015). Parkinson-causing α-synuclein missense mutations shift native tetramers to monomers as a mechanism for disease initiation. Nature Communications, 6, 7314.
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9

Differentiating iPSCs to Brain Microvascular Endothelial Cells

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Human iPSCs IMR90–4 (WiCell, Madison, WI; Yu et al., 2007 ) were maintained and differentiated to brain microvascular endothelial cells (BMECs) based on the protocol developed by Lippmann et al. (2014) (link). Briefly, iPSCs were expanded on Matrigel (Corning Inc., Corning, NY) in mTeSR1 medium (STEMCELL Technologies, Vancouver, BC, Canada) till colonies approached borders of their neighbors. Cells were then switched to a differentiation medium DMEM/F12 medium with HEPES containing 20% KnockOut Serum Replacement, 1× MEM Non-Essential Amino Acids Solution, 1 mM L-glutamine and 0.1 mM β-mercaptoethanol (Sigma, St. Louis, MO). After 6 days of differentiation, cells were switched to Human Endothelial Serum Free Medium supplemented with 1% Platelet Poor Derived Serum (Biomedical Technologies, Baltimore, MD), 20 ng/mL bFGF (R&D Systems, Minneapolis, MN), and 10 μM retinoic acid (Sigma), and cultured for two more days before passaging them for BBB cocultures. All media were replenished daily throughout the culture. All reagents not specified were from Life Technologies (Carlsbad, CA).
Wang Y.I., Abaci H.E, & Shuler M.L. (2016). Microfluidic Blood-Brain Barrier Model Provides In Vivo-Like Barrier Properties for Drug Permeability Screening. Biotechnology and bioengineering, 114(1), 184-194.
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10

Cell Culture Protocol for HeLa, HCT116, and R63 ES Cells

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HeLa (ATCC) and HCT116 [37] (kind gift of R. Farber) cells were maintained in 1 g/L glucose D-MEM (Invitrogen), both supplemented with 10% FBS (Invitrogen) and 1x NEAA (Invitrogen). R63 ES cells [38] (kind gift of H. Wheadon) were maintained on Nunc tissue culture plates (Davidson & Hardy Ltd., Belfast, UK) which were gelatinised with 0.1% porcine gelatin (Sigma-Aldrich, Dorset, UK). Components of ES cell medium are from Invitrogen unless otherwise stated. ES cells were cultured in KnockOut™ D-MEM supplemented with 15% KnockOut™ Serum Replacement, 1% ES-cell qualified fetal bovine serum, 1x NEAA, 2 mM L-glutamine and 0.1 mM β-mercaptoethanol (Sigma-Aldrich). Stable CPEB1 knockdown cells were created using the Knockout™ Single Vector (Clontech, Sainte-Germaine-en-Laye, France) as per the manufacturer’s instructions, and were maintained under the same culture conditions as the parental HeLa cells with the addition of 400 µg/µl geneticin (Invitrogen). All cells were cultured at 37°C, 5% CO2 in a humidified atmosphere.
Rutledge C.E., Lau H.T., Mangan H., Hardy L.L., Sunnotel O., Guo F., MacNicol A.M., Walsh C.P, & Lees-Murdock D.J. (2014). Efficient Translation of Dnmt1 Requires Cytoplasmic Polyadenylation and Musashi Binding Elements. PLoS ONE, 9(2), e88385.
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