Draining LNs were isolated from graft recipients on postoperative day 42, and a single-cell suspension was passed via a 70 μm cell strainer (Corning, CA, USA). Viable single cells were subjected to plate counting as 5 × 105 cells/well in 96-well plates on RPMI media (Welgene Inc., Gyeongsan-si, Republic of Korea) with 1% fetal bovine serum (FBS; Gibco BRL, Karlsruhe, Germany) for 48 h. The cultured single cells were harvested and immunostained with the following antibodies: PE-anti-mouse CD11c antibody (1:100, host:hamster, cat# 117307, BioLegend, San Diego, CA, USA), Alexa Fluor 647-anti-mouse I-Ad antibody (1:100, host:mouse, cat# 115010, BioLegend, San Diego, CA, USA), Alexa Fluor 488-anti-mouse CD4 antibody (1:100, host:mouse, cat# 100423, BioLegend, San Diego, CA, USA), and PE-anti-mouse IFN-gamma antibody (1:100, host:rat, cat# 12-7311-82, Invitrogen, Carlsbad, CA, USA). All antibodies were stained appropriately with the matched isotype controls. The stained cells were analyzed with FACS Melody (BD Biosciences, Franklin Lakes, NJ, USA) and FlowJo software X 10.5.3 (FlowJo LLC, Ashland, OR, USA).
Rpmi media
Manufactured by Welgene
RPMI media is a commonly used cell culture medium formulated to support the growth and maintenance of a variety of cells, including human and mammalian cell lines. It provides the necessary nutrients, vitamins, and other components to sustain cell health and proliferation in a controlled in vitro environment.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Software x 10, by FlowJo (1 mentions)
Pe anti mouse cd11c antibody, by BioLegend (1 mentions)
Alexa fluor 488 anti mouse cd4 antibody, by BioLegend (1 mentions)
Facsmelody, by BD (1 mentions)
70 μm cell strainer, by Corning (1 mentions)
8 hydroxy 2 deoxyguanosine 8 ohdg, by Santa Cruz Biotechnology (1 mentions)
Hacat cells, by Korean Cell Line Bank (1 mentions)
2 7 dichlorofluorescin diacetate dcf da, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin pen strep, by Welgene (1 mentions)
Raw 264.7 cell, by Korean Cell Line Bank (1 mentions)
Powerplex 1, by Promega (1 mentions)
Rpmi 1640, by Welgene (1 mentions)
4 protocols using rpmi media
1
Lymph Node Cell Isolation and Analysis
2
Experimental Breast Cancer Metastasis
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For experimental metastasis, breast cancer cells (4T1) were injected into the tail vein. An infrared lamp was used to dilate the caudal venous vessels. BALB/c mice were injected with 5 × 10³ 4T1 cells in RPMI media (WelGENE, Gyeongsan-si, Korea) to 100 μl final volume per mouse.
3
Oxidative Stress and Inflammation Assays
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GOMV was purchased from Toronto Research Chemicals (North York, ON, Canada). GOMV was dissolved in dimethyl sulfoxide (DMSO) (vehicle) and used in all in vitro experiments at a dilution ratio of 1/1000. LPS and TPA were purchased from Sigma (St. Louis, MO, USA). Dulbelco’s modified Eagle’s media (DMEM), Roswell Park Memorial Institute (RPMI) media, feral bovin serum (FBS), and penicillin/streptomycin (Pen/Strep) were purchased from WELGENE (Daegu, Korea). RAW 264.7 cells and HaCaT cells were purchased from Korean Cell Line Bank (Seoul, Korea). They were maintained in RPMI media supplemented with 10% FBS and Penicillin/Streptomycin (100 U/ml). Polyclonal antibodies against NRF2, total actin, and 8-hydroxy-2′-deoxyguanosine (8-OH-dG) were purchased from Santa Cruz biotechnology (Santa Cruz, CA, USA). 4-hydroxynonenal (4-HNE) antibody was purchased from Abcam (Cambridge, MA, USA). 2′,7′-dichlorofluorescin diacetate (DCF-DA) was purchased from Thermo Fisher Scientific (Waltham, MA, USA).
4
Cell Culture and Authentication Protocol
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Wild-type HCT116 and Staufen1 knockout (KO) HCT116 cells were cultured in RPMI media (Welgene) supplemented with 10% Fetalgro bovine growth serum (RMbio). KG-1 cells were grown in RPMI-1640 (Welgene) supplemented with 10% fetal-bovine serum (Thermo Fisher). All cells were incubated at 37 °C in a humidified atmosphere of 5 % CO2. All cell lines used in this study were male. All cell lines were authenticated using short tandem repeat (STR) profiling(PowerPlex 1.2; Promega), and results were compared with reference STR profiles available through the ATCC.
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