The coding sequence of OsNPF7.6 for subcellular localization was amplified using the pMD19-T vector and ligated into pSAT6A-GFP [33 (link)] with the correct direction. The OsNPF7.6-GFP vector was transformed into Arabidopsis mesophyll protoplasts and identified by a laser scanning microscope (LSM410; Carl Zeiss, Jena, Germany). Plasma membrane dye FMTM4-64FX (Invitrogen, Life Technologies, Carlsbad, CA, USA) was used as marker.
Fmtm 4 64fx
Manufactured by Thermo Fisher Scientific
Sourced in United States
The FMtm 4-64FX is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is a multi-functional device designed for various laboratory applications. The core function of the FMtm 4-64FX is to perform precise measurements and analysis tasks. Further details on the intended use or specific features of this product are not available.
Automatically generated - may contain errors
Lab products found in correlation
Actingreentm 488, by Thermo Fisher Scientific (2 mentions)
Pkh 67 kits, by Merck Group (2 mentions)
Alexa fluor 568, by Proteintech (2 mentions)
Cdc42 inhibitor ml141, by MedChemExpress (2 mentions)
Lipofectaminetm rnaimax, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Proteintech (2 mentions)
Anti ki67, by Cell Signaling Technology (2 mentions)
Calnexin, by Abcam (2 mentions)
Cdc42, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Anti cd9, by Abcam (2 mentions)
Lsm 410, by Zeiss (1 mentions)
Mowiol 4 88, by Polysciences (1 mentions)
Tcs sp8, by Leica camera (1 mentions)
Hybridization oven, by Thermo Fisher Scientific (1 mentions)
Poly l lysine coated slide, by Merck Group (1 mentions)
Axio observer z1, by Zeiss (1 mentions)
Cd105, by Abcam (1 mentions)
Pkh26, by Merck Group (1 mentions)
6 protocols using fmtm 4 64fx
1
Subcellular Localization of OsNPF7.6
2
Detecting MAdCAM-1 Orientation by FRET
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For detecting the orientation of MAdCAM-1 ectodomain relative to cell membrane, CHO-K1/MAdCAM-1 or CHO-K1/MAdCAM-1-Δmucin cells were seeded on poly-L-Lysine (100 μg/ml) coated surface in HBS and incubated for 30 min at 37°C. Adherent cells were treated with HBS or 0.01 U/ml sialidase in HBS for 20 min at 37°C to remove cell surface anionic sialic acid of glycoconjugate. Then cells were fixed with 3.7% paraformaldehyde for 10 min at room temperature. Non-specific sites were blocked by incubation with 10% serum in HBS for 10 min at room temperature. 20 μg/ml Alexa Fluor 488-conjugated 8C1 Fab fragment was used to stain cells for 30 min at 37°C. Then cells were washed twice with HBS and labeled with 10 μM FMTM 4-64FX (Invitrogen) for 1 min on ice. After one wash, cells were immediately mounted with Mowiol® 4-88 (Polysciences) mounting solution under a coverslip. The slides were kept in dark and subjected to photobleach FRET acquisition by a confocal microscope (TCS SP8, Leica). FRET efficiency (E) was calculated as E = 1–(Fdonor(d)Pre/Fdonor(d)Post), where Fdonor(d)Pre and Fdonor(d)Post are the mean donor emission intensity of pre- and post-photobleaching.
3
Membrane Staining Protocol for Live-Cell Imaging
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Membrane staining was performed according to Bigge et al (2023) (link). Cells were treated with 0.5% DMSO or 30 μM BCI for 2 h and then adhered to coverslips for 2 min. Coverslips were moved to ice and 200 μg/ml membrane dye (FMTM 4-64FX, fixable; F34653; Thermo Fisher Scientific) for 1 min, covered, fixed with 4% PFA in 1× HBSS for 10 min, and washed for 3 × 3 min in 1× PBS (Bigge et al, 2023 (link)). Coverslips were air-dried, mounted to slides with Fluoromount-G, and imaged on the spinning disk confocal microscope. For quantification, summed slice projections were created and processed as described in KAP-GFP staining. The area selection measured encompassed an entire cell for 10 cells per image, three images per treatment and time point, across three trials.
4
Fluorescent Staining of Early Division E. faecalis
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Overnight cultures of E. faecalis OG1RF, EFC3C, and EFC3Py were normalized to OD600 = 0.5 in phosphate buffer saline (PBS, pH 7.4). COE1-3C or COE1-3Py was added to the cultures at the following concentrations: wild type E. faecalis OG1RF (1 and 0.5 μM, respectively), EFC3C (1 and 0.5 μM, respectively), and EFC3Py (4 μM of each), incubated for 60 min at 37°C and followed by addition of 10 μg/mL FMTM 4-64FX (ThermoFisher Scientific). After staining, the cells were washed three times in PBS, and 20 μL of cells was spotted onto poly-L -lysine coated slides (Sigma–Aldrich), dried at 46°C in a hybridization oven (ThermoFisher Scientific) for 15 min, mounted with Vectashield mounting medium (Vecalotor Laboratories, Inc.), and imaged using a Zeiss Axio Observer Z1 fitted with a 100×/1.30 oil immersion objective (Carl Zeiss, Göttingen, Germany).
Quantification of fluorescence distribution along the cell was performed using PSICIC (Guberman et al., 2008 (link)). Cells with a perimeter > 4.8 μm and ≤ 8 μm were defined as early division cells (Kandaswamy et al., 2013 (link)) and chosen for the quantitation of the fluorescence of FM4-64 and COEs. Fluorescence intensity along the circumference of the cell was plotted against the cell perimeter coordinates of 1–100 units to generate a fluorescence distribution profile. Quantitative analysis was performed in two independent experiments.
Quantification of fluorescence distribution along the cell was performed using PSICIC (Guberman et al., 2008 (link)). Cells with a perimeter > 4.8 μm and ≤ 8 μm were defined as early division cells (Kandaswamy et al., 2013 (link)) and chosen for the quantitation of the fluorescence of FM4-64 and COEs. Fluorescence intensity along the circumference of the cell was plotted against the cell perimeter coordinates of 1–100 units to generate a fluorescence distribution profile. Quantitative analysis was performed in two independent experiments.
5
Immunofluorescence analysis of Cdc42 in cells
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Anti-CD9, CD63, Alix, calnexin, CD31, CD34, CD45, CD73, CD90 and CD105 antibodies were purchased from Abcam (Cambridge, UK). Anti-Ki-67, RhoA, Rac1, Cdc42 and β-actin antibodies were purchased from Cell Signaling Technology (Danvers, USA). Alexa Fluor 488 and Alexa Fluor 568 secondary antibodies were purchased from Proteintech (Rosemont, IL, USA). Goat anti-rabbit/anti-mouse IgG IRDyel 800cw secondary antibodies were purchased from Abbkine (Redlands, CA, USA). PKH-26 and PKH-67 kits were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lipofectamine TM RNAiMAX, FM TM 4-64FX and ActinGreen TM 488 were purchased from Thermo Fisher (Eugene, Dregon, USA). SiCdc42, Cdc42-EGFP and Cdc42-mCherry fusion protein expression plasmids were purchased from GenePharma (Suzhou, China). Cdc42 inhibitor ML141 was purchased from MedChemExpress (Monmouth Junction, USA).
6
Characterization of Cdc42 Regulators
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Anti-CD9, CD63, Alix, calnexin, and CD31 antibodies were purchased from Abcam (Cambridge, UK). Anti-Ki-67, RhoA, Rac1, Cdc42 and β-actin antibodies were purchased from Cell Signaling Technology (Danvers, USA). Alexa Fluor 488 and Alexa Fluor 568 secondary antibodies were purchased from Proteintech (Rosemont, IL, USA). Goat anti-rabbit/anti-mouse IgG IRDyel 800cw secondary antibodies were purchased from Abbkine (Redlands, CA, USA). PKH-67 kits were purchased from Sigma-Aldrich (St.
Louis, MO, USA). Lipofectamine TM RNAiMAX, FM TM 4-64FX and ActinGreen TM 488 were purchased from Thermo Fisher (Eugene, Dregon, USA). SiCdc42, Cdc42-EGFP and Cdc42-mCherry fusion protein expression plasmids were purchased from GenePharma (Suzhou, China). Cdc42 inhibitor ML141 was purchased from MedChemExpress (Monmouth Junction, USA).
Louis, MO, USA). Lipofectamine TM RNAiMAX, FM TM 4-64FX and ActinGreen TM 488 were purchased from Thermo Fisher (Eugene, Dregon, USA). SiCdc42, Cdc42-EGFP and Cdc42-mCherry fusion protein expression plasmids were purchased from GenePharma (Suzhou, China). Cdc42 inhibitor ML141 was purchased from MedChemExpress (Monmouth Junction, USA).
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