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Rpmi media

Manufactured by Lonza
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Sourced in United States, Switzerland

RPMI media is a cell culture medium primarily used for the growth and maintenance of mammalian cells. It provides the necessary nutrients and components to support the survival and proliferation of various cell types in a laboratory setting.

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Lab products found in correlation

Dnase 1, by Roche (13 mentions) Collagenase type 4, by Merck Group (12 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions) Dmem media, by Lonza (3 mentions) Fetal bovine serum (fbs), by Lonza (3 mentions) Ficoll hypaque, by GE Healthcare (2 mentions) Red blood cell lysis buffer, by Merck Group (2 mentions) Collagenase d, by Roche (2 mentions) Accucheck counting beads, by Thermo Fisher Scientific (2 mentions) Turbofect, by Thermo Fisher Scientific (2 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Fetal bovine serum (fbs), by Gemini Bio (2 mentions) Dnase 1, by Thermo Fisher Scientific (2 mentions) Benzylpenicillin, by Merck Group (1 mentions) Saponin, by Merck Group (1 mentions) Gentamicin, by Sanofi (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) Dimethyl sulfoxide (dmso), by Corning (1 mentions) Human ab serum, by Lonza (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)

34 protocols using rpmi media

1

Isolation and Enumeration of Skin Cells

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Immune cells from debulking surgeries or skin xenografts were isolated, as described previously.42 (link) After harvesting, skin tissues were recovered overnight in RPMI media (Lonza) supplemented with 20% FBS (GeminiBio), 100 mM L-glutamine and ×1 penicillin/streptomycin at 4°C. After that, the tissues were minced into small pieces in 10% FCS-supplemented RPMI, followed by incubation in Collagenase type I (Thermo Fisher, 0.2%) and DNase I (Thermo Fisher, 30 Kunitz Units/mL) at 37°C for 2 h with shaking (350 rpm). Cells were passed through 70 μm cell-strainer, washed and recovered in RPMI media (Lonza) supplemented with 20% FBS, 100 mM L-glutamine and penicillin/streptomycin for 4h or overnight at 37°C. We quantified the total skin cell numbers using fluorescent AccuCheck Counting Beads (Invitrogen) by flow cytometry. Each skin sample had its area calculated and all data were normalized by skin area (in cm2).
Borges T.J., Abarzua P., Gassen R.B., Kollar B., Lima-Filho M., Aoyama B.T., Gluhova D., Clark R.A., Islam S.A., Pomahac B., Murphy G.F., Lian C.G., Talbot S.G, & Riella L.V. (2022). T cell-attracting CCL18 chemokine is a dominant rejection signal during limb transplantation. Cell Reports Medicine, 3(3), 100559.
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2

Quantifying Pneumococcal Internalization in MDM

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MDM were challenged with opsonized S. pneumoniae for 3 h then washed three times in PBS, incubated for 30 min in RPMI media (Lonza) with 40 units/mL of benzylpenicillin (Sigma) and 20 mg/mL gentamicin (Sanofi). The cells were then washed three times in PBS and incubated in 250 μL of 2% saponin (Sigma) for 12 min at 37°C in 5% CO2, then 750 μL of PBS was added, followed by vigorous pipetting. The number of internalised viable bacteria were measured by counting the number of colony forming units on Colombia blood agar (CBA) after 24 h incubation at 37°C in 5% CO2 contained in these lysates measured in triplicate.
Cole J., Angyal A., Emes R.D., Mitchell T.J., Dickman M.J, & Dockrell D.H. (2021). Pneumolysin Is Responsible for Differential Gene Expression and Modifications in the Epigenetic Landscape of Primary Monocyte Derived Macrophages. Frontiers in Immunology, 12, 573266.
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3

Corneal Single-Cell Suspension Preparation

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Corneas were digested to prepare single cell suspensions as described previously [41 (link), 42 (link)]. Briefly, corneal tissues were incubated in RPMI media (Lonza, Walkersville, MD, USA) that contained 2 mg/mL collagenase type IV (Sigma-Aldrich Corp.) and 2 mg/mL DNase I (Roche, Basel, Switzerland) for 45 minutes at 37°C, and then filtered through a 70-μm cell strainer for flow-cytometric analysis.
Bengani L.C., Kobashi H., Ross A.E., Zhai H., Salvador-Culla B., Tulsan R., Kolovou P.E., Mittal S.K., Chauhan S.K., Kohane D.S, & Ciolino J.B. (2020). Steroid-eluting contact lenses for corneal and intraocular inflammation. Acta biomaterialia, 116, 149-161.
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4

Cytotoxicity Assessment of Compounds

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Cytotoxicity (IC50) of compounds 1–5 was determined against seven human cancer cell lines by using an MTT assay according to an established procedure [39 (link)]. The cell lines were cultured in DMEM (Gibco, ThermoFisher Scientific, Hilden, Germany) and RPMI media (Lonza, Cologne, Germany) for MCF-7. All cell lines were supplemented with 10% fetal bovine serum (Gibco) and incubated under 10% CO2 at 37 °C. Epothilone B was used as a positive control and methanol as a negative control.
Primahana G., Risdian C., Mozef T., Sudarman E., Köck M., Wink J, & Stadler M. (2020). Nonocarbolines A–E, β-Carboline Antibiotics Produced by the Rare Actinobacterium Nonomuraea sp. from Indonesia. Antibiotics, 9(3), 126.
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5

Single-Cell Cornea Suspension Protocol

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Single-cell suspensions were prepared from corneas as previously described.11 (link) In brief, corneas were digested in RPMI media (Lonza, Walkersville, MD, USA) containing 2 mg/mL collagenase type IV (Sigma-Aldrich Corp., St. Louis, MO, USA) and 2 mg/mL DNase I (Roche, Basel, Switzerland) for 45 minutes at 37°C and then filtered through a 70-μm cell strainer.
Mittal S.K., Mashaghi A., Amouzegar A., Li M., Foulsham W., Sahu S.K, & Chauhan S.K. (2018). Mesenchymal Stromal Cells Inhibit Neutrophil Effector Functions in a Murine Model of Ocular Inflammation. Investigative Ophthalmology & Visual Science, 59(3), 1191-1198.
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6

Stable Expression of ROR1, ROR2, and PTK7 in BaF3 Cells

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BaF3 cells (DSMZ, #ACC 300) were grown in RPMI media (Lonza, Basel, Switzerland) supplemented with 10% FBS, 50 U/ml penicillin, 50 µg/ml streptomycin, and 10% WEHI conditioned supernatant. For stable expression of human ROR1, ROR2, and PTK7 (or their truncated variants) in BaF3, proteins were cloned into the pEF-IRES-P vector [14 (link)] and transfected using Nucleofection as previously described [3 (link)].
293T cells were cultured in DMEM media (Gibco™, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS, 50 U/ml penicillin, 50 µg/ml streptomycin. Transient transfection of 293T cells was done using TurboFect (Thermo Fisher Scientific) according to the manufacturer’s instructions.
Raivola J., Dini A., Salokas K., Karvonen H., Niininen W., Piki E., Varjosalo M, & Ungureanu D. (2022). New insights into the molecular mechanisms of ROR1, ROR2, and PTK7 signaling from the proteomics and pharmacological modulation of ROR1 interactome. Cellular and Molecular Life Sciences, 79(5), 276.
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7

Isolation and Cryopreservation of PBMCs

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Peripheral blood mononuclear cells (PBMCs) from healthy volunteer donors at the National Institutes of Health (NIH3) blood bank were collected under research protocols approved by the NIH Institutional Review Board (99-CC-0168) using apheresis. Also apheresis samples were obtained from patients treated with moxetumomab pasudotox or SS1P after informed consent in clinical trials (protocols 08-C-0026 and 06-CC-0150). A total of 36 PBMC samples were selected based on previously determined responses to PE38 peptides (25 (link)). Samples were qualified for the study if itdemonstrated a response to one of the peptides described in Table I with all the following criteria: ≥85 spot forming cells (SFC)/106 cells, ≥3-fold the response in the no peptide control, and ≥3% of all SFC.
PBMCs were isolated and cryopreserved as previously described (27 (link)). Briefly, buffy coat was collected after Ficoll-Hypaque (GE Healthcare) density-gradient separation. Cells were washed three times with Dulbecco’s phosphate buffered saline (PBS) without Ca and Mg and cryopreserved in liquid nitrogen at a concentration of 1–3×107 cells/ml in RPMI media (Lonza) supplemented with 10% heat-inactivated human AB serum (Gemini) and 7.5% DMSO (Cellgro).
Mazor R., Addissie S., Jang Y., Tai C.H., Rose J., Hakim F, & Pastan I. (2016). Role of HLA-DP in the Presentation of Epitopes from the Truncated Bacterial PE38 Immunotoxin. The AAPS journal, 19(1), 117-129.
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8

PBMC Isolation and HLA Typing

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Peripheral mononuclear cells (PBMCs) were collected from apheresis samples of 40 naïve donors, four patients that were treated with immunotoxin containing PE38 and five ATL patients under protocols approved by the National Institutes of Health Institutional Review Board (99-CC-0168) and (08-C-0026), respectively with informed consent. Samples were isolated using the gradient-density separation by Ficoll-Hypaque (GE Healthcare) and frozen in liquid nitrogen using 10% human AB serum (Gemini) RPMI media (Lonza) and 7.5% DMSO (Sigma Life Science). Class II HLA typing was performed using PCR sequence-specific primers/sequence-specific oligonucleotides–based tissue typing by the HLA typing unit in the Warren G. Magnuson Clinical Center, NIH or by the Texas BioGene Molecular Typing Laboratory.
Mazor R., Kaplan G., Park D., Jang Y., Lee F., Kreitman R, & Pastan I. (2017). Rational design of low immunogenic anti CD25 recombinant immunotoxin for T cell malignancies by elimination of T cell epitopes in PE38. Cellular immunology, 313, 59-66.
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9

PCa Cell Line Maintenance and Authentication

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The human PCa cell lines LNCaP, VCaP, C4-2, C4-2B, PC3, LAPC, 22Rv1, as well as the human b-lymphocyte cell line WME-099, were either recently purchased (ATCC, in vitro Technologies, Lane Cove, Australia) or authenticated (AGRF, Melbourne, Australia). Cells were maintained in RPMI media (Lonza, Basel, Switzerland) supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA) in a humidified incubator with 5% CO2 at 37 °C.
Ma Y., Luk A., Young F.P., Lynch D., Chua W., Balakrishnar B., de Souza P, & Becker T.M. (2016). Droplet Digital PCR Based Androgen Receptor Variant 7 (AR-V7) Detection from Prostate Cancer Patient Blood Biopsies. International Journal of Molecular Sciences, 17(8), 1264.
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10

CRISPR-Mediated Knockin in U2OS Cells

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U2OS (ATCC HTB-96) cells were grown in RPMI media (Lonza), supplemented with 10% FBS (GIBCO) and 1% penicillin-streptomycin (Invitrogen). For KI cells were transfected with corresponding gRNA and one or two (NeoR and HygroR for mAID-mCherry KI) HR templates. Growth medium was changed 8 h after transfection, 2.5 µM DNAPK inhibitor was added for 48 h. KI cells were selected with G418, puromycin and/or hygromycin until the non-transfected control died, followed by single-cell cloning and KI validation by PCR and/or western blot. Transfections were carried out using Lipofectamine 2000 (Thermofisher), according to manufacturer’s instructions.
293FT cells (Thermofisher #R70007) were grown in DMEM media (Lonza), supplemented with 10% FBS (GIBCO) and 1% penicillin-streptomycin (Invitrogen).
Vipat S., Gupta D., Jonchhe S., Anderspuk H., Rothenberg E, & Moiseeva T.N. (2022). The non-catalytic role of DNA polymerase epsilon in replication initiation in human cells. Nature Communications, 13, 7099.
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