RAW264.7 cells were obtained from China Center for Type Culture Collection (Wuhan, China). All cell lines have been authenticated using STR profiling and tested for mycoplasma contamination. RAW264.7 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum. BMDCs were harvested after 7 days of culture with GM-CSF (20 ng/mL; PeproTech, Rocky Hill, NJ, USA) and IL-4 (10 ng/mL; PeproTech), and BMDMs with M-CSF (50 ng/mL, PeproTech). The purity of BMDCs and BMDMs was more than 90% assessed by flow cytometry. Atg7-knockdown BMDCs (Atg7 KD) were established by siRNA (Ribobio) transfection using riboFECT reagent (Ribobio). LPS (0111:B4, L3024, Sigma-Aldrich), FITC-LPS (0111:B4, F3665, Sigma-Aldrich), z-VAD-fmk (HY-16658B, MedChemExpress), Erastin (E7781, Sigma-Aldrich), Ferrostatin-1 (SML0583, Sigma-Aldrich), Rapamycin (V900930, Sigma-Aldrich), Chloroquine (C6628, Sigma-Aldrich) were supplemented into culture medium respectively in the following experiments.
Fitc lps
Manufactured by Merck Group
Sourced in United States
FITC-LPS is a fluorescent derivative of lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria. It is used in laboratory settings as a tool for the detection and study of LPS-related biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Monolith nt protein labeling kit red nhs, by NanoTemper (3 mentions)
Monolith nt 115, by NanoTemper (3 mentions)
Monolith nt capillaries, by NanoTemper (3 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Lps 0111 b4, by Merck Group (2 mentions)
Peptidoglycan pgn sa, by InvivoGen (2 mentions)
Murnac l ala γ d glu mdap, by InvivoGen (2 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
M csf, by Thermo Fisher Scientific (1 mentions)
Raw264.7 cells, by National Collection of Authenticated Cell Cultures (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Ferrostatin 1, by Merck Group (1 mentions)
Erastin, by Merck Group (1 mentions)
Ribofect reagent, by RiboBio (1 mentions)
Z vad fmk, by MedChemExpress (1 mentions)
Syto9, by Thermo Fisher Scientific (1 mentions)
Guava easycyte, by Merck Group (1 mentions)
22 protocols using fitc lps
1
Establishment and Characterization of Macrophage Cell Models
2
Binding of LPS and WTA in Bacteria
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To detect the binding of LPS from P. aeruginosa to S. aureus, FITC-LPS (Sigma-Aldrich, United States) and free LPS (Sigma-Aldrich, United States) were mixed with 105 CFU of S. aureus. The fluorescence intensity was detected, and the number of S. aureus SA113 bound to FITC-LPS was enumerated with a Guava easyCyte flow cytometer (Merck Millipore). To assess the binding of WTA of S. aureus or free lipoteichoic acids (LTA; Sigma-Aldrich, United States) to P. aeruginosa, mid-log phase S. aureus and P. aeruginosa were stained with hexidium iodide (Thermo Fisher Scientific, Waltham, MA, United States) and SYTO 9 (Invitrogen; Thermo Fisher Scientific) according to the instruction provided by the vendors. Labeled S. aureus and P. aeruginosa were mixed at a ratio of 1:1 and 105 CFU/ml of the bacteria was used for flow cytometry analysis. Five thousand fluorescent-stained cells were counted in a gate set in a hexidium iodide versus SYTO 9 dot plot.
3
Endotoxin Distribution in Pregnant Mice
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In order to observe the distribution of endotoxin in pregnant mice, 20 mg/kg fluorescein isothiocyanate labeled LPS (FITC-LPS, Sigma Company, USA) [19 (link)] was given on the 18th day and performed fluorescence imaging through the small animal in vivo imaging system. The pregnant mice in each group were given FITC-LPS on the 18th day of pregnancy, and fluorescence imaging was performed on the in vivo fluorescence imaging system (LB983, Berto, Germany) after intragastric administration 6–7 h.
4
Lipid-Mediated Gene Delivery Optimization
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Poly(D,L-lactide-co-glycolide) (Resomer® RG 503H, free carboxylic acid, MW 24–38 kD), dichloromethane, polyvinyl alcohol (PVA, MW 30–70 kD, 87–90% hydrolyzed), polyethylenimine (linear, MW 2.5 kD), poloxamer 407, class B CpG ODN 2007 (phosphorothioate backbone modified with a sequence of 5'-TCG TCG TTGTCG TTTTGTCG TT-3' and its non-CpG ODN form, FITC-LPS (derived from Escherichia coli O111:B4) and lipopolysaccharide (LPS) (derived from Escherichia coli O111:B4, TLR ligand tested) were obtained from Sigma-Aldrich (Oakville, ON, Canada). Pam3CSK4 and Rhodamine-Pam3CSK4 (endotoxin level tested) were obtained from InvivoGen (San Diego, CA, USA). Quant-iT™ OliGreen® ssDNA reagent was from Life Technologies.
5
Evaluating LPS and Cytokine Binding of M-Se@SiO2
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To evaluate the LPS- and cytokine-binding ability of M-Se@SiO2, M-Se@SiO2 (1 mg/mL) were mixed with PBS containing 10% FBS and FITC-LPS (Sigma, 100 ng/mL), TNF-α (85 pg/mL), or IL-6 (360 pg/mL) at 37 °C for 30 min. The samples were centrifuged at 16,000 × g for 15 min to remove the nanospheres. LPS remaining in the supernatant was measured as the fluorescence intensity, and cytokines in the supernatant were quantified by ELISA (Anogen, Canada).
6
Molecular Pathways Modulation in Inflammation
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Alginate, LPS, PMB, FITC-phalloidin, FITC-LPS and 4′,6-diamidino-2-phenylindole (DAPI) were obtained from Sigma-Aldrich (St. Louis, MO, USA). RPMI-1640 medium, Dulbecco’s modified Eagle’s medium (DMEM), penicillin and streptomycin were purchased from Hyclone (Logan, UT, USA). Foetal bovine serum (FBS) was obtained from Biological Industries (Beit-Haemek, Israel). Anti-mouse TLR4, MD2, MyD88, F4/80 and Ly6G antibodies were from purchased Abcam (Cambridge, UK). Antibodies against Akt, phosphor-Akt (p-Akt), NF-κB p65, IκB, phosphor-IκB (p-IκB), mTOR, phosphor-mTOR (p-mTOR), p70 S6 kinase (p70 S6K), phosphor-p70 S6K (p-p70 S6K), p38, phosphor-p38 (p-p38), JNK, phosphor-JNK (p-JNK), ERK, phosphor-ERK (p-ERK) and iNOS were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-β-actin, -β-tubulin, -GAPDH and -Lamin B1 antibodies were purchased from Proteintech (Hubei, China). Inhibitors LY 294002, SB 20358, SP 600125 and PD 98059 were obtained from Selleck (Shanghai, China) and TAK-242 and rapamycin from Invitrogen (Carlsbad, CA, USA). The other chemicals were all purchased from Macklin Biochemical Technology (Shanghai, China).
7
Gal8 Modulates LPS Uptake in BMDMs
8
Gal-8 Modulates LPS Binding to CD14 in BMDMs
9
Isolation and Labeling of Bacterial LPS
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LA, A6 and their fragments were obtained from Innovate Biopharmaceuticals, Inc. (Raleigh, NC). LPS from Escherichia coli serotype 0111: B4 labeled with FITC (FITC-LPS) was purchased from Sigma.
10
Investigating EV-LPS Binding and Effects
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10 µg M0φ‐EVs or M2φ‐EVs were mixed with FITC‐LPS (Sigma, USA) according to these different ratios of 10:0.5–10:64 for 1 h at 37°C in PBS, with a final volume of 100 µL (Kumari et al., 2023 (link)). For detection of the binding ability of EVs and LPS by flow cytometry, all the unbound LPS in each mixture was removed by UC at 120,000 × g for 30 min at 4°C, and the pellets were resuspended in PBS for further analysis; for detection of the effect of EV‐bound LPS, the above mixtures of EVs and LPS, LPS or LPS transfection were directly added to MH‐S cells for 24 h. After incubation, the treated cells and supernatant were collected for further analysis.
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