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7 protocols using rpmi 1640 medium

1

PBMC Isolation from Healthy Individuals

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EXAMPLE 7

PBMC isolation. Healthy individuals (negative for HIV, hepatitis B virus, and with or without any HSV infection history) were recruited at the University of California Irvine Institute for Clinical and Translational Science. Between 40 and 100 ml blood was drawn into yellow-top Vacutainer tubes (Becton Dickinson). The serum was isolated and stored at −80° C. for detection of antiHSV-1 and antiHSV-2 Abs, as previously described by Chentoufi, A. et al., Asymptomatic human CD4+ cytotoxic T-cell epitopes identified from herpes simplex virus glycoprotein B. J. Virol. 82: 11792-11802 (2008), the content of which is hereby incorporated by reference in its entirety. PBMCs were isolated by gradient centrifugation using leukocyte separation medium (Cellgro, Manassas, Va.). The cells were washed in PBS and resuspended in complete culture medium consisting of RPMI 1640 medium containing 10% FBS (Gemini Bio-Products, Woodland, Calif.) supplemented with 1× penicillin/L-glutamine/streptomycin, 1× sodium pyruvate, 1× nonessential amino acids, and 50 μM 2-ME (Life Technologies, Rockville, Md.). Aliquots of freshly isolated PBMCs were also cryopreserved in 90% FCS and 10% DMSO in liquid nitrogen.

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2

Asthma Drugs Impact on CCR7 & NKG7

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In order to assess the effects of asthma medications on CCR7 and NKG7 levels, we isolated PBMC from six healthy volunteers consist of three female and three males; All volunteers were Han nationality and the median age was 43years (range 36–48 years). Then, PBMC were cultured in RPMI 1640 medium supplemented with 10% human serum (Gemini Bio-Products) and then treated with 10−5 M dexamethasone for 12 and 24 h. RNA of cell samples was then collected for RT-PCR.
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3

Neutrophil Isolation from Murine Blood

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Peripheral blood was collected via cardiac puncture, anticoagulated with EDTA, and then layered over Ficoll-Histopaque 1077 (Sigma Aldrich). After centrifugation (4°C, 1500 rpm) the supernatant was discarded, and red blood cells were lysed using ACK lysis buffer (GIBCO). After 5 min cold PBS was added and cells were washed twice. The isolated neutrophils were resuspended in RPMI 1640 medium + 2% inactivated mouse serum (Gemini Bio-Products).
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4

Neutrophil Isolation from Murine Blood

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Peripheral blood was collected via cardiac puncture, anticoagulated with EDTA, and then layered over Ficoll-Histopaque 1077 (Sigma Aldrich). After centrifugation (4°C, 1500 rpm) the supernatant was discarded, and red blood cells were lysed using ACK lysis buffer (GIBCO). After 5 min cold PBS was added and cells were washed twice. The isolated neutrophils were resuspended in RPMI 1640 medium + 2% inactivated mouse serum (Gemini Bio-Products).
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5

Culturing C. elegans and HEp-2 Cells

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C. elegans strain N2 was maintained in monoxenic cultures with the addition of Escherichia coli strain OP50, and propagated on nematode growth medium (NGM) agar plates at 25°C as previously described by Brenner (1974 (link)). Human epithelial cells (HEp-2) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). HEp-2 cells were grown in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Gemini Bio-Products, West Sacramento, CA, USA) at 37°C in an atmosphere containing 5% CO2.
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6

Generating Human Myeloma Cell Lines

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Human MM cell lines RPMI8226 and MM.1S were maintained in RPMI-1640 medium with 10% fetal bovine serum (Gemini BioProducts, US), 100 units/mL penicillin, and 100 g/mL streptomycin at 37 °C and 5% CO2. Murine MM cell line 5TGM1 with consistent luciferase gene expression was maintained in the same culture condition. The cell lines were verified by short tandem repeat analysis and tested for mycoplasma contamination. To generate MM cells with a consistently low ALCAM expression, human MM cell lines RPMI8226 and MM.1S were infected with two different ALCAM shRNA lentiviruses (#TLHVU2300, Transomic Tech., US).
The following oligonucleotides were used as shRNA sequences to target ALCAM (sh1 5′-CAGAGGAATCTCCTTATATA-3′ and sh2 5′-CCGAAGGAATAAGAAGCTCAA-3′). Infected cells were selected and maintained in the culture medium with the addition of puromycin (Sigma Aldrich, US). The control viruses were ordered from Transomic Tech (#TLHVU2300, Transomic Tech., US). Human bone marrow stromal cells (BMSCs) were derived and maintained as previously described [12 (link)].
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7

Ovarian and Breast Cancer Cell Lines

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Ovarian cancer cell lines (SKOV3ip1, SKOV3-TR, HeyA8, HeyA8-MDR, A2780, A2780-CP20, IGROV-1, ES2, OVCAR3, and OVCAR5), an ovarian epithelial cell line (HIO-180), and a breast cancer cell line (MDA-231) were cultured as previously described (4 ). In brief, all cell lines were maintained in RPMI 1640 medium that was supplemented with 10% fetal bovine serum and 0.1% gentamicin sulfate (Gemini BioProducts, Calabasas, CA) in 5% CO2 and 95% air at 37°C. Cells were routinely screened for mycoplasma species (GenProbe detection kit; Fisher Scientific, Pittsburgh, PA). All experiments were performed with 70–80% confluent cultures.
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