The HMC3 human microglial cell line (ATCC CRL-3304) and EMEM medium (ATCC 30–2003) were purchased from American Type Culture Collection (ATCC, Manassas, VA). BrdU (59–14-3) and mouse anti-BrdU (B2531) monoclonal antibody (mAb) were purchased from Sigma (St. Louise, MO). BAY 11–7082 was obtained from Cayman Chemical (Ann Arbor, MI, cat# 10010266). Human IL-6 DuoSet ELISA (DY206) kits were purchased from R&D Systems (Minneapolis, MN). Phospho-p53 (Ser 15) antibody (#9284), p53 mouse mAb (#2524), NF-κB p65 rabbit mAb (#8242), p21 Waf1/Cip1 rabbit mAb (#2947), and β-Actin mouse mAb (#3700) were purchased from Cell Signaling (Danvers, MA). Alexa Fluor-594 labeled goat anti-mouse IgG (#A10680) and Alexa Fluor-647 labeled goat anti-rabbit IgG (#A21244) were purchased from Thermo Fisher (Waltham, MA). Mouse anti-human p16 mAb (cat# 51–1325GR) was obtained from BD Biosciences. Mouse anti-human CD68 mAb (Cat# 14–0688-82, clone: KP1) was obtained from Thermo Fisher. Pro-Long Gold Anti-Fade Reagent (#9071) was obtained from Cell Signaling Technology (Danvers, MA).
Hmc3 human microglial cell line
Manufactured by American Type Culture Collection
Sourced in United Kingdom, United States
The HMC3 human microglial cell line is a well-characterized, immortalized primary human microglial cell model derived from the human brain. It maintains key functional properties of primary human microglia and serves as a useful in vitro tool for research on microglial biology and neurological conditions.
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Lab products found in correlation
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Hyclone, by GE Healthcare (2 mentions)
Nf κb p65 rabbit mab, by Cell Signaling Technology (1 mentions)
β actin mouse mab, by Cell Signaling Technology (1 mentions)
Bay 11 7082, by Cayman Chemical (1 mentions)
P21 waf1 cip1 rabbit mab, by Cell Signaling Technology (1 mentions)
Alexafluor 594 labeled goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 labeled goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Cell Signaling Technology (1 mentions)
Emem medium, by American Type Culture Collection (1 mentions)
Pc12 cell line, by American Type Culture Collection (1 mentions)
Af1935, by R&D Systems (1 mentions)
Pa5 16545, by Thermo Fisher Scientific (1 mentions)
Chip kit one step, by Abcam (1 mentions)
6 protocols using hmc3 human microglial cell line
1
Characterizing Human Microglial Cell Responses
2
Culturing HMC3, PC12, and iPSC-derived Microglia
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HMC3 human microglial cell line (CRL-3304) and PC12 cell line (CRL-1721) were purchased from ATCC (American Type Culture Collection). HMC3 cells were cultured in DMEM medium with 10% FBS, 1% l -glutamine, and 1% penicillin/streptomycin (P/S) and maintained in a 37 °C incubator at 5% CO2. PC12 cells were cultured in RPMI-1640 medium supplemented with 10% HS, 5% FBS, and 1% P/S then placed in humidified air chamber containing 5% CO2 at 37 ℃. The monolayer cells were harvested by trypsin and seeded into microplates to assess the compound effect on neurite outgrowth. Human iPSC-derived microglia (Cat#BX-0900) was purchased from BrainXell.
3
Culturing Human Microglial and HEK293 Cells
4
Investigating Transcriptional Regulation of LOX-1
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We identified the binding sites for NF-kB, Oct-1 and HIF-1α in the promoter region of the human OLR1 gene from the UCSC Genome Browser (http://genome.ucsc.edu ). To identify whether the transcription factors NF-κB and HIF-1α control the expression of LOX-1 in microglia, we performed a chromatin immunoprecipitation assay using the ChIP Kit-One Step (ab117138; Abcam, Cambridge, UK) with the HMC3 human microglial cell line (CRL-3304; American Type Culture Collection, Manassas, VA). According to the manufacturer’s instructions, we collected OGD-treated microglial cells, fixed them with 1% formaldehyde and fragmented them with sonication. We used antibodies against NF-κB (PA5-16545; Thermo Fisher Scientific) and HIF-1α (AF1935; R&D Systems) and primer sets for NF-κB and HIF-1α (Additional file 3 : Table S2).
5
Culture of Human Microglial Cells
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The HMC3 human microglial cell line was obtained from American Type Culture Collection (ATCC CRL-3304). HEK293 and HMC3 cells were cultured in Eagle's Modified Minimum Essential Medium (EMEM), ATCC modification (ATCC supplemented with 10% fetal bovine serum, defined (HyClone, GE Healthcare SH30070.03); 50 U/mL penicillin, 50 µg/mL streptomycin (Gibco 22400-089). Cells were maintained at 37°C in a 5% CO2 in air atmosphere.
6
Culturing HMC3 Human Microglial Cells
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The HMC3 human microglial cell line was purchased from the American Type Culture Collection (ATCC, LGC Standards, Manassas, VA, USA; Cat. No. CRL-3304). Cells were cultured in Eagle’s Minimum Essential Medium (EMEM; Merck; Cat. No. M4655) with the addition of 10% fetal bovine serum (FBS; Cat. No. 10500-064; ThermoFisher), sodium pyruvate (1×; ThermoFisher; Cat. No. 11360-070), non-essential amino acids (1×; Cat. No. 11140-035; ThermoFisher) and penicillin (100 U/mL)/streptomycin (100 μg/mL; ThermoFisher) at 37 °C with 5% CO2. Based on experimental plans, cells were plated with the following densities: 600 k cells/well in 6-well plates, 400 k cells/well in 12-well plates, 15 k cells/well in 96-well plates (Falcon, Milan, Italy).
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