Cells were cultured at 37°C incubator with humidified environment and 5% CO2. RAW264.7 cells (FH0328, FuHeng BioLogy, ShangHai, China) were incubated and passaged with DMEM medium (SH30022.01, Cytiva, Pittsburgh, USA) containing 10% FBS (16,140,071, Gibco, Rockville, USA) and 100 μ/mL penicillin–streptomycin‐amphotericin B (C100C8, NCM Biotech, SuZhou, China) before seeding and drug intervention, and the medium was changed every other day. The BMMs were obtained from the fresh femurs of 6‐week‐old C57/BL6 mice as follows. First, the mice sacrificed by anaesthesia were sterilized in 75% ethanol for 5 min. Then the soft tissues of both lower extremities were cleaned, and the femur and tibia were separated rinsed twice with DMEM medium. Next, cut the bone ends with surgical scissors in the ultra‐clean bench, rinse the bone marrow cavity with DMEM and filter it through a 70 μm cell strainer. And add the red blood cell lysate and centrifuge at 1000 r/min for 5 min to remove the supernatant, finally resuspend and culture in DMEM medium with 10% FBS, 50 ng/mL M‐CSF (462‐TEC‐010/CF, R&D Systems, Minneapolis, MN, USA) and 100 μ/mL Penicillin–Streptomycin‐Amphotericin B.
Sh30022
Manufactured by Cytiva
Sourced in United States
SH30022.01 is a laboratory product manufactured by Cytiva. It is designed for general laboratory use.
Automatically generated - may contain errors
Lab products found in correlation
Ab260075, by Abcam (2 mentions)
Sh30023, by Cytiva (2 mentions)
462 tec 010 cf, by R&D Systems (1 mentions)
Net531001mc, by PerkinElmer (1 mentions)
Be13 115e, by Lonza (1 mentions)
F0392, by Merck Group (1 mentions)
Hyclone, by Cytiva (1 mentions)
D9891, by Merck Group (1 mentions)
De17 602e, by Lonza (1 mentions)
Hepes buffer, by Lonza (1 mentions)
Bml ei347, by Enzo Life Sciences (1 mentions)
Sv30160, by Cytiva (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Ab6721, by Abcam (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Interleukin 2 (il 2), by Miltenyi Biotec (1 mentions)
Hydrocortisone, by STEMCELL (1 mentions)
6 protocols using sh30022
1
Culturing and Differentiating Bone Marrow Macrophages
2
Quantifying Glucose Metabolism in Cells
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G55 cells, 786-O-LUC and 786-O-HIF1 cells were plated in pyruvate-free high glucose DMEM supplemented with P/S, HEPES and 10% dialyzed FBS (dFBS, 10,000 Mw cut-off: F0392, Sigma-Aldrich). Then cells were treated with DMOG (0.2 mM) and supplemented as necessary with sodium pyruvate (1 mM: BE13-115E, Lonza) for 24 h. Subsequently, the cells were maintained for 2 h in a 5 mM glucose pyruvate-free DMEM medium (SH30022.01, Cytiva HyClone and 11966025, Gibco) with the corresponding treatments. Then, cell media was replaced with labeling solution (pyruvate-free 5 mM glucose DMEM, without FBS, supplemented with 80 μCi/mmol of 5-3H-Glucose (NET531001MC, PerkinElmer) and the corresponding treatments. Cells were incubated in labeling solution at 37°C for 2 h. Then, the supernatant was transferred to glass vials sealed with rubber stoppers. 3H2O was captured over 48 h at 37°C in hanging wells containing Whatman paper soaked with H2O. Radioactivity was quantified by liquid scintillation counting.
3
Hypoxia Signaling Pathway Regulation
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The 786-O-LUC/HIF1α/HIF1α-bHLH∗,18 (link) G55, MDA-MB-231 and A549 cell lines, and the murine embryonic fibroblasts (MEFs) were all maintained in pyruvate-free Dulbecco’s high glucose modified Eagle’s Medium (DMEM: SH30022.01, Cytiva HyClone) supplemented with 100 units/mL of penicillin and 100 μg/mL streptomycin (P/S: DE17-602E, Lonza), 20 mM HEPES buffer (17-737E, Lonza) and 10% fetal bovine serum (FBS: SV30160.03, Cytiva HyClone). In the experiments, 786-O-LUC/HIF1α/HIF1α-bHLH∗ cells were treated with doxycycline (1 μg/mL: D9891, Sigma-Aldrich) to induce vector expression. Cells were kept at 37°C in an atmosphere with 5% of CO2 and 95% air for normoxic conditions. To induce hypoxia the cells were maintained at 0.5% O2 in an Invivo2 400 workstation (Ruskinn) or exposed to dimethyloxalylglycine (DMOG, 0.1–1 mM: BML-EI347, Enzo Life Sciences, Inc.), a pharmacological inhibitor of prolyl-4-hydroxylase. All experiments were carried out in 5 mM glucose unless specifically stated.
4
Culturing HeLa and Bovine Endometrial Cells
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The human epithelial carcinoma HeLa cell line (ab260075, Abcam) and primary bovine endometrial epithelial cells (BEECs) were respectively cultured in Dulbecco’s modified Eagle’s medium (DMEM)/high glucose (SH30022.01, Cytiva) and DMEM (SH30023.01, Cytiva) supplemented with 10% fetal bovine serum (FBS; 10099141, Gibco) and penicillin/streptomycin (15140122, Gibco) at 37°C and 5% CO2. Primary BEECs were obtained from three Holstein cows for cell culture after sacrifice and were previously isolated, purified, and preserved in our laboratory (44 (link)).
5
Cell Line Cultivation and Antibody Characterization
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NK-92, K562, and MCF-7 cell lines were obtained from ATCC (CRL-2407, CCL-243, and HTB-22 respectively). NK-92 and K562 cells were cultured in RPMI-1640 (Gibco™, Cat# 11875093) supplemented with 10% horse serum (Gibco™, Cat# 16050122), 10% fetal bovine serum (Sigma-Aldrich), 25 mM HEPES pH 7, 100 U/mL penicillin-streptomycin (Gibco™, 10, 000 U/mL Cat# 15140-122), 200 U/mL IL-2 (Miltenyi Biotec), and 1 mM hydrocortisone (Stemcell). MCF-7 cells were cultured in Dulbecco’s Modified Eagle Medium (Cytiva SH30022.01) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 100 U/mL penicillin-streptomycin. Cells were incubated at 37 °C with 5% CO2 and media was changed every 2–3 days.
The antibodies used in this manuscript consisted of α-polySia mAb 735 (BioAspect BA-Ab00240-2.0), α-CD56 (R&D systems, mAB2408), α-beta-tubulin (Abcam, ab6046), streptavidin-HRP (Biolegend, 405210), goat α-mouse IgG-HRP (R&D systems, HAF007), goat α-rabbit IgG-HRP (Abcam, ab6721), and α-QSOX2 (Abcam, ab191168 and ab121376).
The antibodies used in this manuscript consisted of α-polySia mAb 735 (BioAspect BA-Ab00240-2.0), α-CD56 (R&D systems, mAB2408), α-beta-tubulin (Abcam, ab6046), streptavidin-HRP (Biolegend, 405210), goat α-mouse IgG-HRP (R&D systems, HAF007), goat α-rabbit IgG-HRP (Abcam, ab6721), and α-QSOX2 (Abcam, ab191168 and ab121376).
6
Culturing HeLa and Bovine Endometrial Cells
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The human epithelial carcinoma HeLa cell line (ab260075, Abcam) and primary bovine endometrial epithelial cells (BEECs) were respectively cultured in Dulbecco’s modified Eagle’s medium (DMEM)/high glucose (SH30022.01, Cytiva) and DMEM (SH30023.01, Cytiva) supplemented with 10% fetal bovine serum (FBS; 10099141, Gibco) and penicillin/streptomycin (15140122, Gibco) at 37°C and 5% CO2. Primary BEECs were obtained from three Holstein cows for cell culture after sacrifice and were previously isolated, purified, and preserved in our laboratory (44 (link)).
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