To test the effect of the inhibitors on resting PMNs, cells (5 × 106) were incubated in 400 µL of HBSS with different inhibitors or the same dilution of DMSO at 25 °C for 30 and 60 min. Cell-free supernatants were collected and assayed lactoferrin (LTF) and matrix metallopeptidase 9 (MMP9), correlating to the release of secondary and tertiary granules, respectively [10 (link)]. Lactoferrin (Abcam, ab108882, Cambridge, UK) and MMP9 (Abcam, ab100610) were assayed by ELISA. To assay PMN degranulation after chemoattractant stimulation, after treatment with aerobic glycolysis inhibitors and activators for 30 min, including 2-deoxyglucose (2 mM, Sigma, D8375, Kawasaki, Japan), shikonin (1 μM, Sigma, S7576), oleanic acid (OA) (10 μM, Sigma, O5504), serine (5 mM, Sigma, S4500), and FBP (500 µM, Sigma, F6803), or no treatment, PMNs were stimulated with 1 µM fMLP (Sigma, F3506) in HBSS for 20 min (37 °C), followed by assaying for the release of granular markers. Positive degranulation controls were performed by stimulating the same amount of PMNs with fMLP plus 10 µM cytochalasin B (CB) (Sigma, C6762) [11 (link)]. Mouse PMNs from bone marrow were obtained with Percoll to assay the levels of LTF and MMP9 using ELISA (USCN, SEA780Mu, and SEA533Mu, Wuhan, China) after fMLP and PMA (Sigma, P1585) stimulation.
C6762
Manufactured by Merck Group
C6762 is a laboratory equipment product manufactured by Merck Group. It serves as a general-purpose centrifuge tube.
Automatically generated - may contain errors
Lab products found in correlation
D8375, by Merck Group (4 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
D8537, by Merck Group (2 mentions)
G7528, by Merck Group (2 mentions)
P7912, by Merck Group (2 mentions)
Net549, by Merck Group (2 mentions)
Ss255 1, by Merck Group (2 mentions)
Formyl methionyl leucyl phenylalanine (fmlp), by Merck Group (1 mentions)
Ab100610, by Abcam (1 mentions)
Ab108882, by Abcam (1 mentions)
S7576, by Merck Group (1 mentions)
O5504, by Merck Group (1 mentions)
F3506, by Merck Group (1 mentions)
P1585, by Merck Group (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Cytochalasin b, by Merck Group (1 mentions)
Net328a250uc, by PerkinElmer (1 mentions)
I0516, by Merck Group (1 mentions)
G1 plus medium, by Vitrolife (1 mentions)
Hor 272, by ProSpec (1 mentions)
8 protocols using c6762
1
Measuring Neutrophil Granule Release
2
Insulin-Stimulated Glucose Uptake Assay
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C2C12 cells were plated on a 6-well plate coated with 5 ug/ml fibronectin (f1141; Sigma-Aldrich). 42 h after transfection (with Lipofectamine 2000), cells were washed in warm PBS, starved in DMEM/0.1% BSA for 6 h, and washed again. After treatment with 200 nM insulin/PBS (I0516; Sigma-Aldrich) for 30 min at 37°C, cells were washed and incubated for 10 min at 37°C with 1 uCi/ml 2-deoxy-d -[3H]glucose (NET328A250UC; PerkinElmer) and 0.1 mM cold 2-deoxy-d -glucose (D8375; Sigma-Aldrich). 20 nM of cytochalasin B (glucose transport inhibitor; C6762; Sigma-Aldrich) was added to control wells. Then, cells were washed in cold PBS, lysed in 0.2 N NaOH for 2 h at room temperature, and radioactivity was determined using scintillation fluid.
3
2-DG Uptake Assay in Keratinocytes
4
Mouse Oocyte Microinjection Protocol
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ICSI was performed using standard techniques as previously described (57 (link)). Briefly, female KM mice were first injected with 5 IU of equine chorionic gonadotropin (CG) (HOR-272, ProSpec). After 48 hours, the female mice were injected with 5 IU of human CG (M2530, Easycheck) again to acquire MII-arrested oocytes. MII-arrested oocytes were incubated with Chatot-Ziomek-Bavister medium (M2750, Easycheck) at 37.5°C in an incubator with 5% CO2. Mouse spermatozoa from cauda epididymides were incubated in HTF (human tubal fluid) medium (M1150, Easycheck). After freezing and thawing, a single sperm head was obtained and microinjected into an MII oocyte using a NIKON inverted microscope and a Piezo device (PrimeTech) in Whitten’s-Hepes medium containing 0.01% polyvinyl alcohol (12360-038, Gibco) and cytochalasin B (3.5 μg/ml; C-6762, Sigma-Aldrich). Once the injection was completed successfully, the oocytes were transferred into G1-Plus medium (10132, Vitrolife) at 37.5°C in an incubator with 5% CO2 in air.
5
Cell Signaling Modulation Assay
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HGF was used at 50 ng/ml final concentration on starved cells (H5691; 20 µg/ml in PBS stock; Sigma-Aldrich). Cytochalasin B was used at 10 µM final concentration (10 mg/ml in DMSO stock; C6762; Sigma-Aldrich). Specific Src family inhibitor PP1 (10 mM in DMSO stock; 567809; Merck Chemicals) or specific FAK inhibitor PF228 (10 mM in DMSO stock; PZ0117; Sigma-Aldrich) were used at a final concentration of 25, 75, and 10 µM, respectively. LiCl solution was used at 30 mM final concentration (105679; Merck Chemicals).
6
Cytochalasin B-Induced Binucleation Assay
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Cells were cultured on autoclaved coverslips and the culture medium was supplemented with cytochalasin B (2 μg/ml; micro-filament assembly inhibitor; C6762, Sigma Aldrich) for 16 hours before the cell harvest to block cytokinesis. The cells were fixed with 4% (v/v) formaldehyde for 15 min at RT in the dark, and after several washes with 1× PBS, cells were counterstained with DAPI (1 μg/ml) in distilled water and mounted using Fluoromount-G (Invitrogen). Images were captured with a Leica DM6B fluorescent microscope at ×63 magnification, and the percentage of binucleated cells with micronuclei was determined. At least 250 binucleated cells were analyzed in each experiment.
7
2-DG Uptake Assay in Keratinocytes
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2-DG uptake assays were performed as previously described49 (link). Primary mouse or human keratinocytes were seeded in triplicate in 12-well plates overnight (50000), washed twice with PBS (Sigma, D8537), incubated in basic KSFM (without any supplementation) or serum free DMEM/F12 medium (Thermo, 11320033, for SCCT8 cells) for 2 hours, washed twice with KRHP buffer, and incubated in 0.45 ml KRHP buffer to each well, and starved for 30 minutes. For inhibition of glucose transporter, Cytochalasin B (10 µM, Sigma, C6762) and phloretin (100 µM, Sigma, P7912) were added to the KRHP medium for another 15 minutes. Uptake was initiated by adding 1µCi 3H 2-DG (25–30 Ci/mmol, PerkinElmer, NET549) and 0.1 mM unlabeled 2-DG (Sigma, D8375) in KRHP to each well for 10 or 20 min. Transport activity was terminated by the rapid removal of the uptake medium and washing 3 times with cold PBS with 25 mM glucose (Sigma, G7528). Cells were lysed with 0.5 ml of 0.5 M NaOH (Fisher Scientific, SS255-1) and neutralized with 0.5 ml or 0.5 M HCl (Sigma, 320331), mixed well. 250 µl of the lysate was transferred to a scintillation vial with scintillation solution, and the sample was quantitated by liquid scintillation counting. Protein concentrations were determined by BCA assay (Thermo, 23227).
8
Mechanism of GBS Internalization in Caco-2 Cells
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To study the mechanisms of GBS internalization, Caco-2 cells were treated with the following chemical inhibitors: chlorpromazine (CPZ; C8138-54, Sigma-Aldrich) and monodasylcadaverine (MDC; D4008, Sigma-Aldrich) for inhibition of clathrin-mediated endocytosis; methyl-β-cyclodextrin (MβDC; C4555, Sigma-Aldrich) and genistein (Gen; G6649, Sigma-Aldrich) for inhibition of cholesterol and tyrosine kinase, respectively, in lipid rafts; cytochalasin B (CytB; C6762, Sigma-Aldrich) and nocodazole (Ndz; M1404, Sigma-Aldrich), for inhibiting cellular actin and microtubules, respectively. Cells were pre-incubated with these compounds for 1 h prior to infection at the indicated concentrations. All infections were performed in presence of inhibitors, while control cells were incubated with vehicle only. Epithelial cell viability was evaluated by trypan blue staining after incubation with the inhibitors and was always > 90%.
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