Idmem

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom, United States

The IDMEM is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is designed to provide a controlled environment for cell culture and tissue engineering applications. The IDMEM maintains a stable temperature, humidity, and atmospheric conditions to support the growth and development of cells and tissues.

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7 protocols using idmem

Stable HEK293 Cell Line Generation

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HEK293 cells grown in IDMEM (Invitrogen, Carlsbad, California, USA) and 10% FBS (Thermo Scientific, Waltham, Massachusetts, USA) were transfected with pLenti-6.3-myc-NT51A WT or variant vectors, pDM2D, and pΔ874 (4ug DNA total) using Lipofectamine 2000 (Invitrogen) and Opti-MEM (Invitrogen).
Lentivirus was harvested four times over 72 hours after transfection and used to infect HEK293s for 24 hours. Infected HEK293s were maintained in IDMEM +10% FBS and 5.5 ug/ml blasticidin S HCL (Invitrogen) throughout subsequent assays. Confirmation of stable overexpression by Western blot is described in detail in the Supplementary Methods.

Saliba J., Zabriskie R., Ghosh R., Powell B.C., Hicks S., Kimmel M., Meng Q., Ritter D.I., Wheeler D.A., Gibbs R.A., Tsai F.T, & Plon S.E. (2016). Pharmacogenetic characterization of naturally occurring germline NT5C1A variants to chemotherapeutic nucleoside analogs. Pharmacogenetics and genomics, 26(6), 271-279.

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Prostate Cancer Cell Line Characterization

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The PC3 human PCa cell line (NCI-DTP Cat# PC-3, RRID:CVCL_0035) is derived from a bone metastasis of a primary prostate adenocarcinoma (ATCC® CRL-1435™) and the DU145 cell line (NCI-DTP Cat# DU-145, RRID:CVCL_0105) from a central nervous system metastasis of a primary prostate adenocarcinoma [27] (link). When transplanted into nude or severe combined immunodeficient (SCID) mice, the cells lines are tumorigenic [28] (link). PCa cell lines were maintained in IDMEM (Gibco, Grand Island, NY) with 10% fetal bovine serum (Gibco), penicillin (500 μg/ml), and streptomycin sulfate (500 μg/ml) at 37 °C in humidified air with 5% CO₂. All cell lines were validated by the STR profiling and showed >85% match. All populations tested negative for the presence of mycoplasma and murine viruses. For each experiment, triplicates were set up and repeated at a minimal of three times.

Sun B.L., Sun X., Casanova N., Garcia A.N., Oita R., Algotar A.M., Camp S.M., Hernon V.R., Gregory T., Cress A.E, & Garcia J.G. (2020). Role of secreted extracellular nicotinamide phosphoribosyltransferase (eNAMPT) in prostate cancer progression: Novel biomarker and therapeutic target. EBioMedicine, 61, 103059.

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Differentiation of HL-60 and PC12 Cells

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HL-60 cells (a human promyelocytic leukemia cell line) were cultured in Iscove's modified Dulbecco's minimum essential medium (IDMEM; Gibco Ltd, Paisley, UK) with 20% FBS and 1% penicillin-streptomycin (PS), and differentiated into PMN-like cells by adding 1.3% DMSO for 96 h as previously reported 24 (link). PC12 (a neuron-like rat pheochromocytoma cell line) neuronal cells were maintained in RPMI 1640 medium (Corning Cellgro, Mediatech, USA) supplemented with 10% FBS and 1% PS and 5% nonessential amino acids. Cells were maintained in an incubator in a controlled atmosphere of 37 °C, 95% relative humidity and 5% CO₂.

Zhang C., Ling C.L., Pang L., Wang Q., Liu J.X., Wang B.S., Liang J.M., Guo Y.Z., Qin J, & Wang J.X. (2017). Direct Macromolecular Drug Delivery to Cerebral Ischemia Area using Neutrophil-Mediated Nanoparticles. Theranostics, 7(13), 3260-3275.

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Isolation and Culture of Primary Microglia

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Primary microglia were obtained from mixed cultures prepared from the cerebral cortex of mice at the postnatal day 1 (P1-3). Microglia cells were isolated by shaking flasks for 45 min at 230 rpm at day 10 after plating. Cells were then seeded on poly-L-ornithine (Sigma) pre-coated wells at the density of 1,5×10⁵ cell/mL in DMEM containing 20 % heat-inactivated fetal bovine serum (FBS) and incubated at 37 ° C in a humidified atmosphere of 5 % CO2 and 95 % air. Where indicated, cells were pre-treated for 45 min with protease inhibitors and EDTA 2.5 mM (Roche) which were directly added to the cell culture medium. N9 cells were maintained in IDMEM (Gibco Laboratories, USA), supplemented with 10 % FBS. For siRNA transfection, N9 cells were plated at a concentration of at 1×10⁵ cells/mL into either 96 or 24 multiwell plate. Specific siRNAs were diluted at a final concentration of 20 nM siRNA. Cells were used within 48–72 h after transfection. When not differently indicated, for in vitro experiments microglia were treated with H-Aβ42 400 nM.

Mazzitelli S., Filipello F., Rasile M., Lauranzano E., Starvaggi-Cucuzza C., Tamborini M., Pozzi D., Barajon I., Giorgino T., Natalello A, & Matteoli M. (2016). Amyloid-β 1–24 C-terminal truncated fragment promotes amyloid-β 1–42 aggregate formation in the healthy brain. Acta Neuropathologica Communications, 4, 110.

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Culturing Neuronal and Leukemia Cell Lines

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The PC12 cell line (a neuron-like rat pheochromocytoma cell line) and the HL-60 cell line (the human promyelocytic leukemia cell line) were purchased from the Cell Bank at the Chinese Academy of Sciences (Shanghai, China). HL-60 cells were cultured in Iscove's modified Dulbecco minimum essential medium (IDMEM; Gibco Ltd., Paisley, UK) with 10% FBS and 1% penicillin–streptomycin, and differentiated into PMN-like cells by adding 1.3% DMSO for 96 h as previously reported²¹ (link). Cells were maintained in an incubator at a controlled atmosphere of 37 °C, 95% relative humidity and 5% CO₂.
PC12 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% (v/v) heat-inactivated fetal bovine serum (FBS), l-glutamine (2 mmol/L), 100 units/mL penicillin, and 100 μg/mL streptomycin in a humidified atmosphere containing 5% CO₂ at 37 °C. The medium was changed every 2–3 days.

Chen B., Luo M., Liang J., Zhang C., Gao C., Wang J., Wang J., Li Y., Xu D., Liu L., Zhang N., Chen H, & Qin J. (2017). Surface modification of PGP for a neutrophil–nanoparticle co-vehicle to enhance the anti-depressant effect of baicalein. Acta Pharmaceutica Sinica. B, 8(1), 64-73.

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Cell Line Maintenance and Authentication Protocol

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PNT1A, LAPC-4, 22RV1, PZ-HPV7, RWPE-1, and HEK293 were purchased from American Type Culture Collection (ATCC, Manassas, VA) and were maintained in medium according to ATCC instruction. C4-2 was originally generated by our lab. In addition, LN95 cell was provided by Dr. Jun Luo (Johns Hopkins Medical School) and maintained in Phenol red-free RPMI 1640 (Life Technologies, Carlsbad, CA) with 10% Charcoal-stripped Fetal bovine serum (CS-FBS). C4-2 and PNTA1 were maintained in RPMI 1640 (Life Technologies) supplemented with 10% FBS. LAPC-4 was cultured in Iscove’s-Dulbecco’s Modified Eagle’s Medium (I-DMEM; Life Technologies) with 10% FBS. VCaP was generously provided by Dr. Ralf Kittler (UT Southwestern Medical Center) and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Life Technologies) containing 10% FBS. Cell lines were passaged in our laboratory for fewer than 40 times after resuscitation. Cell lines were authenticated using AmpFLSTR®Identifier® PCR Amplification kit (Life Technologies) and confirmed as mycoplasma-free using MycoAlert® kit (Lonza Walkersville, Inc. Walkersville, MD). All the antibodies used in this study were purchased from several commercial sources listed in Supplemental Table S1.

Wang B., Lo U.G., Wu K., Kapur P., Liu X., Huang J., Chen W., Hernandez E., Santoyo J., Ma S.H., Pong R.C., He D., Cheng Y.Q, & Hsieh J.T. (2017). Developing new targeting strategy for androgen receptor variants in castration resistant prostate cancer. International journal of cancer, 141(10), 2121-2130.

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Isolation of Intestinal Immune Cells

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Small intestinal and colonic lamina propria mononuclear cells were isolated as previously described66 (link). Briefly, Peyer’s Patches were removed, and whole small intestine and colon were opened longitudinally, cut into pieces and incubated for 30 minutes in cold calcium- and magnesium-free PBS containing 30 mM EDTA, washed extensively, and incubated for several rounds in iDMEM (Life Technologies) containing collagenase D (0.7 mg/ml; Roche) and Liberase TL (2.5 mg/ml; Roche) at 37 °C. The remaining intestinal fragments were collected and pressed through 100μm filters (BD Biosciences), mixed with the collected supernatants. Mononuclear cells were separated by a 40/80% (wt/vol) Percoll (Sigma-Aldrich) density gradient and washed prior to staining for flow cytometry.
For cytokine quantification, either single-cell suspensions or isolated CD4⁺ T cells from mLNs by a negative magnetic separation (MACS) using CD4⁺ T cell isolation kit (Miltenyi Biotech) were cultured on plate bound α-CD3ε (clone: 145-2C11) and α-CD28 (clone: 37.51) (eBioscience) (both at 2.5 μg/ml) for 72 hours.

Coutaz M., Hurrell B.P., Auderset F., Wang H., Siegert S., Eberl G., Ho P.C., Radtke F, & Tacchini-Cottier F. (2016). Notch regulates Th17 differentiation and controls trafficking of IL-17 and metabolic regulators within Th17 cells in a context-dependent manner. Scientific Reports, 6, 39117.

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