Ficoll pm400

Manufactured by GE Healthcare

Sourced in United States, Sweden

Ficoll PM400 is a high-molecular-weight, non-ionic, synthetic polymer. It is commonly used in biomedical research and clinical applications for the separation and purification of cells, proteins, and other biological macromolecules.

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Lab products found in correlation

Rnaseout, by Thermo Fisher Scientific (3 mentions) Superase in, by Thermo Fisher Scientific (3 mentions) Ficoll pm70, by GE Healthcare (3 mentions) Protein a sepharose beads, by Merck Group (2 mentions) Allprep dna rna micro kit, by Qiagen (1 mentions) Gentamicin, by Fujifilm (1 mentions) Nanoject 2 injection apparatus, by Drummond (1 mentions) Trizol reagent, by Thermo Fisher Scientific (1 mentions) Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions) Lin28, by Proteintech (1 mentions) U2af2, by Santa Cruz Biotechnology (1 mentions) Sf3b1, by Santa Cruz Biotechnology (1 mentions) Srsf1, by Santa Cruz Biotechnology (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Hepes, by Merck Group (1 mentions) Collagenase p, by Roche (1 mentions) Cellsens microscope imaging software, by Olympus (1 mentions) Phenol chloroform isoamyl alcohol, by Merck Group (1 mentions) Dithiothreitol (dtt), by Merck Group (1 mentions) Np 40, by Roche (1 mentions)

Close spelling variants of this product

Ficoll (495 citations)

16 protocols using ficoll pm400

Preparation and Characterization of SM-PEG Conjugates

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SM-PEG₆ (succinimidyl-((N-maleimidopropionamidol)-hexethylene
glycol) ester) was
obtained from Thermo Scientific (1 g) or Pierce (100 mg) (Rockford,
IL). SM-PEG₄₅ was supplied by Nanocs (New York, NY). SM-PEG₇₀ was supplied by NOF American Corporation. Ficoll PM400 was
purchased as a spray-dried powder from GE Healthcare (Pittsburgh,
PA). Sulfo-N-hydroxysuccinimide acetate was from
Thermo Fisher. All other chemicals were at least A.C.S. grade purchased
from Sigma-Aldrich (St. Louis, MO) or USP grade purchased from either
Sigma or J.T. Baker (Avantor, Center Valley, PA).

Milley B., Kiwan R., Ott G.S., Calacsan C., Kachura M., Campbell J.D., Kanzler H, & Coffman R.L. (2016). Optimization, Production, and Characterization of a CpG-Oligonucleotide-Ficoll Conjugate Nanoparticle Adjuvant for Enhanced Immunogenicity of Anthrax Protective Antigen. Bioconjugate Chemistry, 27(5), 1293-1304.

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Injection and Maturation of Xenopus Oocytes

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A 1:1 mixture of anti-XSSH IgG and anti-XSSH IgG-NLS and constitutively active S3A chick cofilin (Nagaoka et al., 1996 (link)) were used for injection. Injection volumes from a pressure injector (CIJ-1; Shimadzu, Kyoto, Japan) were calibrated by measuring the volume of an aqueous drop delivered into mineral oil. Injections were given in OR2 medium containing 5% Ficoll PM400 (GE Healthcare) by inserting a glass needle into the marginal zone of an oocyte in a volume of 20–30 nl/oocyte. After injection, the injected oocytes were incubated in OR2 medium for at least 2 h at 16°C and then treated with progesterone to induce oocyte maturation at 19°C. Phalloidin injection was carried out as described elsewhere (Okada et al., 2012 (link)).

Yamagishi Y, & Abe H. (2015). Reorganization of actin filaments by ADF/cofilin is involved in formation of microtubule structures during Xenopus oocyte maturation. Molecular Biology of the Cell, 26(24), 4387-4400.

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In vitro Fertilization and Microinjection of Xenopus Embryos

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X. laevis eggs were collected and in vitro-fertilized with wild-type or transgenic sperm, then cultured and/or microinjected by standard procedures (Sive et al., 2000 ). In vitro fertilization with p27::GFP [Xla.Tg(Xtr.cdknx:GFP)^Papal, RRID:EXRC_0043] (Carruthers et al., 2003 (link)) frozen sperm was performed by NXR protocol. Frozen sperm was removed from liquid nitrogen and immediately swirled in a 37°C water bath for 30sec, then resuspended in 500μl room-temperature 1/3x Modified Frog Ringer’s solution (MR) by using a micropestle. Sperm mixture was then added to the clutch of eggs and mixed into the monolayer for 3–5min, then flooded with 1/3x MR. After fertilization, embryos were injected with mRNAs and DNAs at the four-cell stage using a PicoSpritzer setup in 1/3x MR with 2.5% Ficoll PM 400 (GE Healthcare, #17–0300-50), and were transferred after injection into 1/3x MR containing 50μg/ml Gentamycin. Drop size was calibrated to about 7–8nl per injection.

Tasca A., Helmstädter M., Brislinger M., Haas M., Mitchell B, & Walentek P. (2021). Notch signaling induces either apoptosis or cell fate change in multiciliated cells during mucociliary tissue remodeling. Developmental cell, 56(4), 525-539.e6.

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Generation of Transgenic Tg Frogs

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To produce Tg frogs, we used the I-SceI meganuclease- [18] (link) and transposon-mediated gene trap [19] (link). Fertilized eggs were injected with I-SceI meganuclease (NEB) and the I-SceI-cleaved plasmid encoding Z-AR and V5, and the Tol II mRNA [19] (link) and the prrT2ARG plasmid encoding Z-AR and GFP, respectively, using a NANOJECT II injection apparatus (Drummond). Tg embryos were cultured in 0.1×MMR with 6% Ficoll PM400 (GE Healthcare) and 50 µg/ml gentamicin (Wako), developed to St. 20 at 18°C, and then transferred to water at room temperature. The tadpoles were continuously reared in water with or without T (50 ng/ml; 150 nM). To confirm the exogenous Z-AR integration into genomic DNA, we extracted DNA from the tail tip of all Tg frogs just after metamorphosis, using the AllPrep DNA/RNA Micro Kit (QIAGEN). The PCR primers used were: forward, 5′-GCGGTTTTTCCAACTTACCA-3′ and reverse, 5′-CGAGACCGAGGAGAGGGTTA-3′).

Fujii J., Kodama M., Oike A., Matsuo Y., Min M.S., Hasebe T., Ishizuya-Oka A., Kawakami K, & Nakamura M. (2014). Involvement of Androgen Receptor in Sex Determination in an Amphibian Species. PLoS ONE, 9(5), e93655.

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Isolation of Hepatic Stellate and Kupffer Cells

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Isolation of HSCs and KCs from rat liver was performed as described previously with modification [28 (link)]. Briefly, rat liver was treated with in situ pronase/collagenase perfusion and subsequently digested in vitro. Next, separation of HSCs and KCs from other hepatic cell populations was achieved by a density gradient-based protocol using two different concentrations of Ficoll PM400 (GE Healthcare, Chicago, IL, USA).

Ohara M., Ohnishi S., Hosono H., Yamamoto K., Yuyama K., Nakamura H., Fu Q., Maehara O., Suda G, & Sakamoto N. (2018). Extracellular Vesicles from Amnion-Derived Mesenchymal Stem Cells Ameliorate Hepatic Inflammation and Fibrosis in Rats. Stem Cells International, 2018, 3212643.

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Cross-linking-free RNA Immunoprecipitation with Molecular Crowders

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Cross-linking-free RNA immunoprecipitation was performed as previously detailed (27 (link)), with the addition of a cocktail of molecular crowders [2.5 mg/ml Ficoll PM400, 7.5 mg/ml Ficoll PM70 (both GE Healthcare) and 250 ng/ml Dextran Sulphate 670k (Fluka)] to the immunoprecipitation step. Briefly, 10 μl of antibody [LIN28 (ProteinTech) or U1 (Santa Cruz)] was incubated with BSA-blocked Protein G sepharose beads (Sigma) overnight, washed five times with NT2 buffer, and resuspended in 850 μl of NT2 buffer with molecular buffer cocktail and 200 U of RNase Out and 100 U of SUPERase IN (Life Technologies). Human ESC cultures that were 50–70% confluent (1–5 × 10⁶ cells) were then washed with phosphate buffered saline (Invitrogen) and lysed with 500 μl of polysome lysis buffer. Nuclei were removed by centrifugation at 5000 × g for 5 min. 100 μl of the cytoplasmic lysate was then added to 900 μl of antibody-coated bead slurry from above for each sample, and rotated at 4°C overnight. The next day, beads were washed five times with NT2, and the RNA eluted with Trizol reagent (Invitrogen), followed by standard RNA precipitation protocols. The RNA was resuspended in 50 μl of RNase-free water and ready for either real-time PCR or microarray analysis, where volumes were kept constant across all samples. 10 μl of RNA was used for each sample for preparation of cRNA for microarray analysis.

Tan S.M., Altschuler G., Zhao T.Y., Ang H.S., Yang H., Lim B., Vardy L., Hide W., Thomson A.M, & Lareu R.R. (2014). Divergent LIN28-mRNA associations result in translational suppression upon the initiation of differentiation. Nucleic Acids Research, 42(12), 7997-8007.

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Salmonella Motility Assay in Viscous Media

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Salmonella strains, SJW1103B (only expressing FljB) and SJW1103 (only expressing FliC), were pre-cultured in 5 mL of Luria–Bertani broth (LB, 1% (w/v) tryptone, 0.5% (w/v) yeast extract, 0.5% (w/v) NaCl) with overnight shaking at 37 °C. Bacterial growth was measured via optical density at 600 nm (OD₆₀₀).
A 5 µL measure of the culture medium was inoculated into 5 mL of fresh LB, and it was incubated for 6 h at 37 °C with shaking. The cells were diluted in the motility buffer (10 mM potassium phosphate pH 7.0, 0.1 mM EDTA, 10 mM sodium D-lactate). The viscosity of the motility buffer was adjusted by adding Ficoll PM400 (GE healthcare, Chicago, IL, USA) to a final concentration of 5%, 10%, or 15%. Swimming motility was observed using a bright-field microscope, CX-41 (Olympus, Tokyo, Japan) with a 40× objective (PlanC N 40× NA 0.65 Olympus) and a 1.25× variable magnification lens (U-CA 1.5 × Olympus) and recorded using a high-speed camera (GEV-B0620M-TC000 IMPREX, Boca Raton, FL, USA). The swimming speed was calculated using the software Move-tr2/2D (Library Co., Tokyo, Japan).

Yamaguchi T., Toma S., Terahara N., Miyata T., Ashihara M., Minamino T., Namba K, & Kato T. (2020). Structural and Functional Comparison of Salmonella Flagellar Filaments Composed of FljB and FliC. Biomolecules, 10(2), 246.

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RIP-ChIP Protocol for Protein-RNA Complexes

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The protocol was adapted from the RIP–ChIP protocol described previously^{45 (link)}. Briefly, protein A Sepharose beads (Sigma) were coated with 3 µg of U1 snRNP, SF3B1, SRSF1, U2AF2 or mouse IgG antibody (Santa Cruz), followed by incubation with 2 mg of Hep3B or SNU398 total cell lysates overnight. The RNA–protein–bead complexes were washed once with NT2 crowders (25 mg Ficoll PM400 (GE Healthcare), 75 mg Ficoll PM70 (GE Healthcare) and 2.5 mg dextran sulfate (Fluka) in 10 ml of NT2 buffer) and five times with NT2 buffer (50 mM Tris pH 7.0, 150 mM NaCl, 1 mM MgCl₂ and 0.05% (v/v) NP-40). Protein–RNA complexes were collected in 100 μl of NET2 buffer (1 mM DTT, 16.7 mM EDTA, 200 U RNaseOUT (Thermo Fisher) and 100 U SUPERase In (Ambion) in 1× NT2 crowder), supplemented with 100 μl of 2× SDS–TE (100 mM Tris pH 7.5, 10 mM EDTA pH 8.0 and 1% SDS). RNA was isolated using TRIzol reagent and subsequently purified with phenol:chloroform:isoamyl alcohol (25:24:1) and chloroform:isoamyl alcohol (24:1).

Chan J.J., Zhang B., Chew X.H., Salhi A., Kwok Z.H., Lim C.Y., Desi N., Subramaniam N., Siemens A., Kinanti T., Ong S., Sanchez-Mejias A., Ly P.T., An O., Sundar R., Fan X., Wang S., Siew B.E., Lee K.C., Chong C.S., Lieske B., Cheong W.K., Goh Y., Fam W.N., Ooi M.G., Koh B.T., Iyer S.G., Ling W.H., Chen J., Yoong B.K., Chanwat R., Bonney G.K., Goh B.K., Zhai W., Fullwood M.J., Wang W., Tan K.K., Chng W.J., Dan Y.Y., Pitt J.J., Roca X., Guccione E., Vardy L.A., Chen L., Gao X., Chow P.K., Yang H, & Tay Y. (2022). Pan-cancer pervasive upregulation of 3′ UTR splicing drives tumourigenesis. Nature Cell Biology, 24(6), 928-939.

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Metacyclic Promastigote Purification

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EP and LP promastigote cultures were prepared as described above and maintained at stationary phase culture for 3 days when cell density, acidification and nutrition depletion trigger the differentiation from procyclic to metacyclic promastigotes. Parasites were collected and adjusted to 3x10⁸ cells/ml. Ficoll PM400 (GE Healthcare) was used to prepare a 20% stock solution in PBS and diluted for preparation of 10% and 5% Ficoll solutions. Four ml of 10% Ficoll were overlaid by 4 ml of 5% Ficoll and 4 ml of parasite suspension were layered on top of the Ficoll cushion. Tubes were centrifuged at 1,300 x g for 15 min at room temperature without brake. The metacyclic-enriched fractions were recovered at the interface between the 10% and 5% Ficoll layers. Parasites were washed with PBS and adjusted to the final concentration required for a given experiment.

Piel L., Rajan K.S., Bussotti G., Varet H., Legendre R., Proux C., Douché T., Giai-Gianetto Q., Chaze T., Cokelaer T., Vojtkova B., Gordon-Bar N., Doniger T., Cohen-Chalamish S., Rengaraj P., Besse C., Boland A., Sadlova J., Deleuze J.F., Matondo M., Unger R., Volf P., Michaeli S., Pescher P, & Späth G.F. (2022). Experimental evolution links post-transcriptional regulation to Leishmania fitness gain. PLoS Pathogens, 18(3), e1010375.

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Xenopus Embryo Manipulation and Assays

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X. laevis eggs were obtained, fertilized, and staged as previously described^{47 (link),48}. Embryos were cultured in 0.33

\times

Modified Ringer (MR) until the mid-blastula transition, after which transferred to 0.1

\times

MR until harvest. Embryo injection was performed using HARVARD Injector in 0.33

\times

MR containing 4% Ficoll-PM400 (GE Healthcare) at indicated stages. Injected embryos were cultured in 4% Ficoll-400 in 0.33

\times

MR before the mid-blastula transition. For the axis duplication assay, mRNA or morpholino was injected into the ventral vegetal region at the 4-cell stage, and the embryos were cultured until the tailbud stage (Fig. 3c)^{49 (link)}. In the animal cap explant induction experiment, animal caps were dissected at stage 8 and cultured until the sibling embryos reached stage 10.5 (Fig. 3d)^{49 (link)}.

Wang Y., Han W., Yun S, & Han J. (2023). Identification of protein phosphatase 4 catalytic subunit as a Wnt promoting factor in pan-cancer and Xenopus early embryogenesis. Scientific Reports, 13, 10240.

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