Rpmi 1640

Manufactured by BioLegend

Sourced in United States

RPMI-1640 is a widely used cell culture medium formulated to support the growth and maintenance of various cell types, including human and animal cells. It provides a balanced set of nutrients, vitamins, and salts essential for cell proliferation and survival.

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Lab products found in correlation

Penicillin streptomycin, by BioLegend (3 mentions) M csf, by BioLegend (1 mentions) Cxcl12, by BioLegend (1 mentions) Cxcl11, by BioLegend (1 mentions) Facscanto 2, by BD (1 mentions) Cd11b m1 70, by Thermo Fisher Scientific (1 mentions) Ebio17b7, by Thermo Fisher Scientific (1 mentions) F4 80 bm8, by BioLegend (1 mentions) Jes5 16e3, by Thermo Fisher Scientific (1 mentions) Rorγt afkjs 9, by Thermo Fisher Scientific (1 mentions) Rm4 5, by BioLegend (1 mentions) Stat3 124h6, by Cell Signaling Technology (1 mentions) Ifn γ xmg1.2, by BioLegend (1 mentions) Mcf 7 cells, by Solarbio (1 mentions) Ir140, by Merck Group (1 mentions) Hela cells, by National Collection of Authenticated Cell Cultures (1 mentions) Ir806, by Merck Group (1 mentions) Zncl2, by Merck Group (1 mentions) Bovine serum albumin (bsa), by Solarbio (1 mentions) Cxcl2, by BioLegend (1 mentions)

10 protocols using rpmi 1640

Isolation and Culture of Murine Macrophages

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HeLa cells, HEK293T cells, RAW264.7 cells, and MEFs were maintained in DMEM (nacalai tesque) with 10% fetal bovine serum (FBS), 1% Penicillin/Streptomycin (nacalai tesque), and 100 µM 2-Mercaptoethanol (nacalai tesque). Mycoplasma contamination was routinely tested and found negative.
For the preparation of bone marrow-derived macrophages (BMDMs), bone marrow cells were cultured in RPMI-1640 (nacalai tesque) with 10% FBS, 1% Penicillin/Streptomycin, 100 μM 2-mercaptoethanol, and 20 ng/ml of macrophage colony-stimulating factor (M-CSF) (BioLegend) for 6 days.
For the preparation of thioglycolate-elicited peritoneal exudate cells (PECs), mice were intraperitoneally injected with 2 ml of 4% (w/v) Brewer’s thioglycollate medium. 3.5 days after the injection, peritoneal macrophages were collected and cultured in RPMI-1640 with 10% FBS, 1% Penicillin/Streptomycin, and 100 μM 2-mercaptoethanol.

Akaki K., Ogata K., Yamauchi Y., Iwai N., Tse K.M., Hia F., Mochizuki A., Ishihama Y., Mino T, & Takeuchi O. (2021). IRAK1-dependent Regnase-1-14-3-3 complex formation controls Regnase-1-mediated mRNA decay. eLife, 10, e71966.

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Transwell Assay for CLL Cell Migration

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Peripheral blood mononuclear cells (PBMC) were isolated using a Ficoll density gradient centrifugation; only samples containing more than 70% CLL cells in PBMC were chosen for the assay. Transmigration of CLL cells was assessed using polycarbonate Transwell inserts with 5-μm pore size (Corning Costar). Briefly, the cells at 1 × 10exp 6/mL were applied to the upper chamber in RPMI-1640 containing 1% bovine serum albumin (BSA) in the presence or absence of CXCL11 (BioLegend). Filters were transferred into the lower wells containing RPMI-1640 with 1% BSA in the presence or absence of CXCL12 (BioLegend). After 3 hours at 37°C in 5% CO₂, cells that migrated into the lower chambers were counted and analysed for CXCR3 and CXCR4 expression on BD FACSCanto II.

Manukyan G., Papajik T., Mikulkova Z., Urbanova R., Kraiczova V.S., Savara J., Kudelka M., Turcsanyi P, & Kriegova E. (2020). High CXCR3 on Leukemic Cells Distinguishes IgHVmut from IgHVunmut in Chronic Lymphocytic Leukemia: Evidence from CD5high and CD5low Clones. Journal of Immunology Research, 2020, 7084268.

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Comprehensive Immune Cell Profiling

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Anti-mouse phosphor-STAT3 (EP2147Y, Abcam), phosphor-NF-κB (pp65) (sc-52401, Santa), phosphor-JNK (pJNK)(9H8), phosphor-ERK (pERK)(197G2), STAT3 (124H6, Cell Signaling), NF-κBp65 (sc8008, Santa), JNK (SC), ERK(137F5), and β-actin (sc-47778, Santa) were purchased. FITC-, PE-, APC- APC/cy7-, PerCP/Cy5.5-, or PE/cy7-conjugated anti-mouse CD4 (RM4-5, Biolegend), CD8 (ZUT270.5, Biolegend), CD45.1 (30-F11, Biolegend), MHCII (I-A/I-E, M5/114.115.2, Biolegend), CD11c (MCA1441GA, Biolegend), CD11b (M1/70, eBioscience), F4/80 (BM8, Biolegend), CD206 (CO68C2), Gr-1(RB6-8C5, eBioscience), IL-10 (JES5-16E3, eBioscience), Foxp3 (MF23, eBioscience), IL-22 (140301, R&D system), IFNγ (XMG1.2, Biolegend), RORγt (AFKJS-9, eBioscience), IL-17A (eBio17B7, eBioscience), and CD3ζ (H146-968, Abcam) antibodies were purchased. Anti- REG3γ (PA517, Thermo) and anti-MUC2 (H300, Santa) were also purchased. FITC-, PE-, APC- APC/cy7-, PerCP/Cy5.5-, or PE/cy7-conjugated isotypic controls were from Biolegend. RPMI1640, DMEM, fetal bovine serum (FBS), and antibiotics were obtained from HyClone. Alexa fluor 488-conjugated goat anti-rabbit IgGH&L and Alexa fluor 594-conjugated goat anti-rabbit IgGH&L (Abcam) were also purchased. 7-AAD was from Abcam.
Lactobacillus reuteri (ATCC PTA 4659) was a gift of BioGaaia, Sweden.

Huang Y., Qi H., Zhang Z., Wang E., Yun H., Yan H., Su X., Liu Y., Tang Z., Gao Y., Shang W., Zhou J., Wang T., Che Y., Zhang Y, & Yang R. (2017). Gut REG3γ-Associated Lactobacillus Induces Anti-inflammatory Macrophages to Maintain Adipose Tissue Homeostasis. Frontiers in Immunology, 8, 1063.

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Multimodal Imaging Probe Synthesis

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ZHO was purchased from Bachem UK Ltd. ICG, IR 806, IR 140, RhB, ZnCl₂, methanol were purchased from Sigma-Aldrich Inc. BSA was purchased from Solarbio Biotechnology Co. Ltd. MCF-7 cells (catalog number SCSP-669S), 4T1 cells (catalog number SCSP-5056), CT26 cells (catalog number TCM37) and Hela cells (catalog number SCSP-504) were provided by the National Collection of Authenticated Cell Cultures. Dulbecco’s Modified Eagle’s Medium (DMEM), Roswell Park Memorial Institute 1640 (RPMI 1640), heat-inactivated fetal bovine serum (FBS), Dulbecco’s phosphate-buffered saline (PBS), trypsin-EDTA, and penicillin-streptomycin were purchased from BioLegend Co. Other materials were purchased from Beijing Chemical Co. Ltd. unless otherwise noted.

Li S., Chang R., Zhao L., Xing R., van Hest J.C, & Yan X. (2023). Two-photon nanoprobes based on bioorganic nanoarchitectonics with a photo-oxidation enhanced emission mechanism. Nature Communications, 14, 5227.

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Neutrophil Migration Assay Protocol

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Neutrophil migration was assessed using 24-well microchambers and polycarbonate filters (5 μm pore size) (Corning, NY, USA) as described previously^{40 (link),64 (link)}. In brief, CD45⁺ CD11b⁺ Ly6G⁺ neutrophils sorted from mouse bone marrow cells were placed in the upper wells (1 × 10⁵ cells/well) of Transwell chambers, and 600 µL of RPMI 1640 medium with or without 100 ng/ml CXCL2 (BioLegend) were added to the lower wells. For migration assays, cells were incubated for 60 min at 37 °C. After the incubation, cells that migrated to the bottom part of the membrane and the lower wells was stained with Hoechst 33,342. The numbers of cells per field were counted in 10 randomly selected visual fields under a microscope (BZ-X810, Keyence, Osaka, Japan), and the mean estimate for individual samples was calculated.

Watanabe A., Koike H., Kumagami N., Shimba S., Manabe I, & Oishi Y. (2023). Arntl deficiency in myeloid cells reduces neutrophil recruitment and delays skeletal muscle repair. Scientific Reports, 13, 6747.

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Expansion of EBV-specific T cells

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Blood samples were obtained from two EBV-seropositive healthy donors with informed consent under the approval of the Ethical Committee for Epidemiology of Hiroshima University. PBMCs were isolated from buffy coats with density centrifugation using lymphocyte separation medium 1077 (Takara Bio, Shiga, Japan). FACS-sorted CD4⁺ T cells or CD8⁺ T cells were plated at 1 × 10⁵ cells per well in a U-bottom 96-well plate and co-cultured with autologous LCL irradiated at 30 Gy starting at 40:1 responder to stimulator ratio in RPMI-1640 supplemented with 10% FCS, 1% Penicillin-streptomycin, and 10 ng/mL human recombinant IL-7 (BioLegend, San Diego, CA, USA). An amount of 10 ng/mL IL-2 was added from day 2 of co-culture. Cells were then re-stimulated with irradiated autologous LCL at a ratio of 1:1 responder to stimulator weekly for a total of 5 stimulation rounds. Thereafter, T cells were expanded in T25 culture flasks on a weekly basis counting, and fed/split as necessary to give a final density of 1–2 × 10⁶ T cells/cm². All cell counts were given as viable cells via trypan blue exclusion of dead cells.

Tamura Y., Yamane K., Kawano Y., Bullinger L., Wirtz T., Weber T., Sander S., Ohki S., Kitajima Y., Okada S., Rajewsky K, & Yasuda T. (2022). Concomitant Cytotoxic Effector Differentiation of CD4+ and CD8+ T Cells in Response to EBV-Infected B Cells. Cancers, 14(17), 4118.

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Isolation and Activation of Cardiac Immune Cells

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To obtain CD4⁺ T lymphocytes and antigen-presenting cells (APCs), single-cell suspensions of splenocytes were sorted using BD FACSAria II (BD Biosciences, San Jose, CA, USA). CD4⁺ T cells and APCs were co-cultured in a 10:1 ratio. CD4⁺ T cells were activated with anti-CD3/CD28 beads (ThermoFisher, USA) or 10 ng/mL of α-MyHC (Caslo), and the conditioned medium was collected on Day 3 for further experiments. Experiments were performed on primary cardiac microvascular endothelial cells (cMVECs) isolated from hearts of 4-week-old Tnf^+/+ and Tnf^−/− mice as described previously.^{19 (link)} cMVECs were cultured in RPMI 1640 supplemented with 10% foetal bovine serum (FBS) and 2 ng/mL recombinant mouse epidermal growth factor (BioLegend, USA). cMVECs were activated with recombinant mouse TNF-α (5 ng/mL, BioLegend) or conditional medium. A detailed description is provided in the Supplemental Methods section.

Rolski F., Tkacz K., Węglarczyk K., Kwiatkowski G., Pelczar P., Jaźwa-Kusior A., Bar A., Kuster G.M., Chłopicki S., Siedlar M., Kania G, & Błyszczuk P. (2023). TNF-α protects from exacerbated myocarditis and cardiac death by suppressing expansion of activated heart-reactive CD4+ T cells. Cardiovascular Research, 120(1), 82-94.

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Isolation and Visualization of Human T and NK Cells

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Human healthy donor peripheral blood mononuclear cells (PBMCs) were isolated using Lymphocyte Separation Solution (Nacalai tesque, Kyoto, Japan). Human T cells and NK cells were enriched from PBMCs by cell depletion of B cells and monocytes using anti-CD19, and anti-CD14 conjugated magnetic beads with Magnisort technology (ThermoFisher). To facilitate visualization, purified T cells were stained with 2 µM Calcein-AM (Nacalai tesque) in PBS for 30 min at 37 °C followed by co-culture. Isolated cells were maintained in a T cell medium; RPMI1640 supplemented with 10% FBS, 100 U/mL human IL-2 and 10 ng/mL human IL-7 (both from Biolegend, CA, USA).

Kabashima A., Matsuo Y., Ito S., Akiyama Y., Ishii T., Shimada S., Masamune A., Tanabe M, & Tanaka S. (2022). cGAS-STING signaling encourages immune cell overcoming of fibroblast barricades in pancreatic cancer. Scientific Reports, 12, 10466.

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Craniotomy-associated Leukocyte Immunosuppression

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The ability of craniotomy-associated leukocyte populations to inhibit polyclonal T cell activation was performed as previously described (20 ). Briefly, leukocyte populations were isolated from the brain, galea, and bone flap by FACS at days 3 and 7 post-infection using the markers for each cell population described in the Flow Cytometry section. CD4⁺ T cells were isolated from the spleens of naïve mice using a CD4⁺ T cell isolation kit (Miltenyi Biotec, Auburn, CA) and immediately labeled with eFluor 670 cell proliferation dye (eBioscience), according to the manufacturer’s instructions. Labeled CD4⁺ T cells were plated at 10⁵ cells/well in a 96-well round-bottom plate in RPMI-1640 supplemented with 10% FBS and 100 ng/mL IL-2 (BioLegend). FACS-purified leukocytes were added at 1:1 ratios to CD4⁺ T cells that were subjected to polyclonal stimulation with CD3/CD28 Dynabeads (Life Technologies), since TCR immunodominant epitopes for S. aureus are not defined. Cells were incubated for 72 h, whereupon the extent of T cell proliferation was determined by flow cytometry using a BD LSR II (RRID:SCR_002159).

Aldrich A.L., Horn C.M., Heim C.E., Korshoj L.E, & Kielian T. (2021). Transcriptional diversity and niche-specific distribution of leukocyte populations during Staphylococcus aureus craniotomy-associated biofilm infection. Journal of immunology (Baltimore, Md. : 1950), 206(4), 751-765.

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Single-Cell Isolation from Solid Tumors and Spleens

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Tumor tissue was digested in the RPMI 1640 medium (Gibco) containing DNAse (60 µg/ml), hyaluronidase (0.2 µl/ml), Collagenase IA and IV (0.2 mg/ml each) for 30 min at 37 °C. Required enzymes were obtained from Sigma. Cell solutions were subsequently washed with RPMI and filtered using a 40 -µm cell strainer to obtain single-cell preparations.
Splenocytes were released from spleens by passing through a 40 -µm cell strainer and washed with RPMI 1640 (Gibco). Erythrocytes were lysed by adding 1x EBC lysis buffer (BioLegend) for 5 min at 4 °C, and cells were subsequently washed with RPMI 1640. Cells were resuspended in the RPMI 1640 medium supplemented with 2% FCS, 100 U/mL streptomycin, 100 mg/mL penicillin, 1% MEM with nonessential amino acids (×100 solution, Gibco) ß-mercaptoethanol (50 µM, Sigma), and sodium pyruvate (1 mM, Gibco), and kept at 37 °C and 5% CO₂ overnight during peptide stimulation.
Erythrocytes were removed from blood samples by adding ×1 EBC lysis buffer (BioLegend) and incubated for 3 min at 4 °C. Cells were subsequently washed with RPMI 1640 (Gibco).

Niemann J., Woller N., Brooks J., Fleischmann-Mundt B., Martin N.T., Kloos A., Knocke S., Ernst A.M., Manns M.P., Kubicka S., Wirth T.C., Gerardy-Schahn R, & Kühnel F. (2019). Molecular retargeting of antibodies converts immune defense against oncolytic viruses into cancer immunotherapy. Nature Communications, 10, 3236.

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