Lo flo medium

Manufactured by Formedium

Sourced in United Kingdom

Lo-Flo medium is a laboratory equipment product designed for low-flow applications. It provides a consistent and controlled flow rate for various experimental and research purposes.

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Lab products found in correlation

Bsa coated 50mm glass bottom dishes, by MatTek (3 mentions) Airyscan confocal microscope, by Zeiss (2 mentions) Hl5 medium, by Formedium (2 mentions) Glass bottom 96 well plate, by Greiner (1 mentions) Blasticidin s, by Thermo Fisher Scientific (1 mentions) Geneticin, by Merck Group (1 mentions) Hygromycin b, by Merck Group (1 mentions) Cellcarrier, by PerkinElmer (1 mentions) Fluostar optima, by BMG Labtech (1 mentions) Em ccd c9100 13, by Hamamatsu Photonics (1 mentions)

Close spelling variants of this product

Lo-Flo (6 citations)

11 protocols using lo flo medium

Phagosome Proteolysis and Lysosomal Activity Assay

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Phagosome proteolysis was determined based on the dequenching of DQ-BSA/Alexa594 conjugated 3μm silica beads (Kisker Biotech) as described (Sattler et al., 2013) . Cells were seeded at 3 x 10 5 per well of a glass-bottom 96 well-plate (Greiner) in Lo-Flo medium (Formedium) in triplicate. After 1 hour, 1.5 x 10 4 reporter beads were added to each well except controls and plates were spun at 800 x g for 10 seconds to synchronize bead uptake.
Cells were then washed twice with Lo-Flo to remove unengulfed beads before fluorescence measurements at 480/510nm and 560/620nm using a plate reader every 2 minutes. After subtraction of background fluorescence, DQ-BSA Signal was normalised for bead uptake using the Alexa594 signal.
Total proteolytic activity was performed in a similar way by adding the same reporter beads to cell lysates (Buckley et al., 2019) . 4 x 10 7 cells/mL in 150mM potassium acetate pH4.0 were lysed by two freeze-thaw cycles in liquid nitrogen. Cell debris was removed by centrifugation at 18,000 x g for 5 minutes at 4°C before 1.23 x 10 5 beads were added 100µL of supernatant in 96 well plates and measured as above. Cathepsin D levels were measured by Western blot using the previously published antibody (Journet et al., 1999) .

Starling G.P., Phillips B.A., Ganesh S., & King J. (2022). Katnip is required to maintain microtubule function and lysosomal delivery to autophagosomes and phagosomes.

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Chemotaxis Assay for Cell Migration

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Cellular morphology, pseudopod dynamics and cell migration were measured by an under-agarose folate chemotaxis assay as described earlier [13] . In brief, 0.4% SeamKem GTG agarose was dissolved in boiling Lo-Flo medium (Formedium).
After cooling, 10 µM folic acid was added. 5 ml of agarose-folate mix was poured into the 1% BSA-coated 50mm glass bottom dishes (MatTek). A 5 mm wide trough was cut with sterile scalpel and filled with 200 µl of 2x 10 6 cells/ml. Cell migration was imaged after 4-6h with 10x and 60x DIC. To examine the localization of labelled proteins in the pseudopods, cells were also imaged using an AiryScan confocal microscope (Zeiss).

Singh S.P., Thomason P.A., & Insall R.H. (2021). Extracellular signalling modulates Scar/WAVE complex activity through Abi phosphorylation.

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Imaging Multinucleate Dictyostelium Cells

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Cells were cultivated in Petri dishes containing nutrient medium [36 (link)] supplemented with 10 µg/mL of blasticidin S (Gibco, Life Technologies Corporation, Grand Island, NY, USA), 10 µg/mL of geneticin (Sigma-Aldrich, St. Louis, MO, USA), or 33 µg/mL hygromycin B (EMD Millipore Corp., Billerica, MA, USA) at 21 ± 2 °C.
For imaging, cells rinsed off the Petri dish were transferred to an HCl-cleaned cover-glass bottom dish (FluoroDish, WPI INC., Sarasota, FL, USA) and kept for 1 to 2 h in LoFlo medium (ForMedium Ltd., Norfolk, UK). The rate of mitosis could be increased by incubating the cells for about 20 h at 4 °C in Petri dishes with nutrient medium and subsequently bringing them to room temperature before transfer to LoFlo medium.
Large cells with wild-type AX2-214 background were produced by electric pulse-induced fusion as described by Gerisch et al. [29 (link)]. Myosin-II-null cells were cultivated in shaken suspension in nutrient medium for about 36 h to get large multinucleate cells [20 (link)]. Multinucleate cells were transferred onto HCl-cleaned cover-glass bottom dishes and incubated in LoFlo medium for about 1 h before imaging was started. Where indicated in the figure legends, cells were overlaid with a thin agarose sheet [37 (link),38 ]. The velocity of unilateral furrow propagation was measured beginning at 1 µm of ingression.

Bindl J., Molnar E.S., Ecke M., Prassler J., Müller-Taubenberger A, & Gerisch G. (2020). Unilateral Cleavage Furrows in Multinucleate Cells. Cells, 9(6), 1493.

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Multinucleate Septase-null Cell Cultivation

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Cells were cultivated in nutrient medium as described by Malchow et al.^{24 (link)}, supplemented with 10 µg/ml of Blasticidin S (B10) (Gibco, Life Technologies Corporation, Grand Island, NY, USA), 10 µg/ml of Geneticin (G10) (Sigma-Aldrich, St. Louis, MO, USA), and/or of 33 µg/ml of Hygromycin B (H33) (Calbiochem, Merck KGaA, Darmstadt, Germany) in plastic Petri dishes at 21 ± 2 °C.
To increase the rate of mitotic stages in the multinucleate Septase-null cells, the nutrient medium was removed and the cells were rinsed off the Petri dishes with Loflo medium (ForMedium Ltd., Norfolk, UK). The cells were then transferred to HCl-cleaned cover-glass bottom dishes (FluoroDish, WPI INC., Sarasota, FL, USA) and either incubated overnight at 21 ± 2 °C in Loflo medium or imaged the same day after incubation for 1–2 h in Loflo medium. Before imaging, cells were overlaid by a thin agarose sheet²⁵ that had been incubated in Loflo medium.

Prassler J., Ecke M, & Gerisch G. (2024). Microtubule dependent sorting of actin-binding proteins in mitosis. Scientific Reports, 14, 11250.

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Pseudopod Formation and Cell Migration

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Formation of pseudopods and cell migration were measured by under-agarose folate chemotaxis assay as described in Laevsky and Knecht, 2003 [59 (link)]. In brief, 0.4% SeamKem GTG agarose in Lo-Flo medium (ForMedium) was prepared by boiling. After cooling, 10 μM folic acid was added. Next, 5 ml of agarose-folate mix was poured into the BSA-coated 50-mm glass-bottom dishes (MatTek). A 5-mm-wide trough was cut with sterile scalpel and filled with 2×10⁶ cells/ml. Cell migration was imaged after 4–6 hours with 10× and 60× DIC. To examine the localization of labeled proteins in the pseudopods, cells were also imaged by AiryScan confocal microscope.

Singh S.P., Thomason P.A., Lilla S., Schaks M., Tang Q., Goode B.L., Machesky L.M., Rottner K, & Insall R.H. (2020). Cell–substrate adhesion drives Scar/WAVE activation and phosphorylation by a Ste20-family kinase, which controls pseudopod lifetime. PLoS Biology, 18(8), e3000774.

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Cultivation and Imaging of Dictyostelium

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D. discoideum AX2 cells were used as an axenic strain and NC4 cells were used as nonaxenic, wild-type cells. The PI3 kinase quintuple knockout strain HM1200 (Hoeller and Kay, 2007 (link)) was obtained from the Dicty Stock Center. For axenic growth, cells were cultivated on Petri dishes under HL5 medium (ForMedium). Nonaxenic cultivation was done on Petri dishes under SorMC buffer supplemented with live Klebsiella aerogenes at an OD₆₀₀ of 2. SorMC buffer consists of 15 mM KH₂PO₄, 2 mM Na₂HPO₄, 50 µM MgCl₂, and 50 µM CaCl₂, pH 6.0. To reduce autofluorescence of axenically cultivated cells in preparation for fluorescence microscopy, cells were incubated overnight in LoFlo medium (ForMedium). For microscopy, cells were put on a 3.5-cm acid-washed glass-bottom MatTek dish.

Veltman D.M., Lemieux M.G., Knecht D.A, & Insall R.H. (2014). PIP3-dependent macropinocytosis is incompatible with chemotaxis. The Journal of Cell Biology, 204(4), 497-505.

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Infection Assay of Legionella in Acanthamoeba

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A. castellanii amoebae were cultured in PYG medium and passaged the day prior to infection such that 2×10⁴ cells were present in each well of a 96-well plate (Cell Carrier, black, transparent bottom from Perkin-Elmer). Cultures of L. pneumophila harbouring the GFP-producing plasmid pNT-28 were resuspended from plates to a starting OD₆₀₀ of 0.1 in AYE medium, and grown overnight on a rotating wheel at 37°C to an OD₆₀₀ of 3. Bacteria were diluted in LoFlo medium (ForMedium) such that each well contained 8×10⁵ bacteria, an MOI of 20. Infections were synchronised by centrifugation at 1500 rpm for 10 minutes. Infected cultures were incubated in a 30°C incubator and the GFP fluorescence was measured by a plate spectrophotometer at appropriate intervals (Optima FluoStar, BMG Labtech) [88] (link). Time courses were constructed and data from the point directly after entry up to stationary phase were used to determine the effect of compounds versus vehicle control.

Kicka S., Trofimov V., Harrison C., Ouertatani-Sakouhi H., McKinney J., Scapozza L., Hilbi H., Cosson P, & Soldati T. (2014). Establishment and Validation of Whole-Cell Based Fluorescence Assays to Identify Anti-Mycobacterial Compounds Using the Acanthamoeba castellanii - Mycobacterium marinum Host-Pathogen System. PLoS ONE, 9(1), e87834.

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Analyzing Cell Migration via Folate Chemotaxis

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Cellular morphology, pseudopod dynamics and cell migration were measured by an under-agarose folate chemotaxis assay as described previously (Singh et al., 2020 (link); Singh and Insall, 2022 (link)). In brief, 0.4% SeamKem GTG agarose in Lo-Flo medium (Formedium) was boiled. After cooling, folic acid to a final concentration of 10 μM was added. 5 ml of agarose-folate mix was poured into the 1% BSA-coated 50 mm glass bottom dishes (MatTek). A 5 mm wide well was cut with a sterile scalpel and filled with 200 μL of 2 × 10⁶ cells/ml. Cell migration was imaged after 4–6 h with 10x and 60x differential interference contrast (DIC) microscopy. To examine the localization of labelled proteins in the pseudopods, cells were also imaged by AiryScan confocal microscopy.

Singh S.P., Paschke P., Tweedy L, & Insall R.H. (2022). AKT and SGK kinases regulate cell migration by altering Scar/WAVE complex activation and Arp2/3 complex recruitment. Frontiers in Molecular Biosciences, 9, 965921.

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Cell Imaging Protocol for Transcription Dynamics

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Cell culture preparation for imaging was carried using cells grown in HL5 medium (FORMEDIUM) supplemented with penicillin+streptomycin. 20 µg/ml G418 selection was added 72 hr after thawing frozen stocks. 18 hr prior to imaging, cells were split into imaging chambers (NUNC LabTek-II) at the appropriate density for imaging the next day at around 20% confluency, drug selection was removed and HL5 was replaced with imaging medium (75% LoFlo medium (Formedium), 10% FBS, 15% HL5). Imaging media was refreshed 1.5 hr before imaging. Imaging was performed using an UltraView Vox spinning disc confocal microscope. Objective, laser lines, camera (Hamamatsu C9100-13 EM-CCD) settings, laser powers and exposure times were optimized to minimize photobleaching and ensure negligible photo-toxicity (measured in terms of the average transcription spot intensity - in trials we found transcription spots were attenuated before reduced cell motility or cell rounding is observed). Data were analysed using custom-built software integrating both cell tracking and spot detection. Code can be accessed at http://www.ucl.ac.uk/lmcb/sites/default/files/Corrigan2016MatlabFiles.zip.

Corrigan A.M., Tunnacliffe E., Cannon D, & Chubb J.R. (2016). A continuum model of transcriptional bursting. eLife, 5, e13051.

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Quantifying Phagosome Proteolysis in Cells

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Phagosome proteolysis was determined based on the dequenching of DQ-BSA/Alexa594 conjugated 3-μm silica beads (Kisker Biotech) as described by Sattler et al. (2013 (link)). Cells were seeded at 3 × 10⁵ per well of a glass-bottomed 96-well plate (Greiner) in Lo-Flo medium (Formedium) in triplicate. After 1 h, 1.5 × 10⁴ reporter beads were added to each well except controls, and plates were spun at 800 × g for 10 s to synchronize bead uptake. Cells were then washed twice with Lo-Flo to remove unengulfed beads before fluorescence measurements at 480/510 nm and 560/620 nm using a plate reader every 2 min. After subtraction of background fluorescence, DQ-BSA Signal was normalized for bead uptake using the Alexa594 signal.
Total proteolytic activity was performed in a similar way by adding the same reporter beads to cell lysates (Buckley et al., 2019 (link)). Samples of 4 × 10⁷ cells/mL in 150 mM potassium acetate at pH 4.0 were lysed by two freeze–thaw cycles in liquid nitrogen. Cell debris was removed by centrifugation at 18,000 × g for 5 min at 4°C before 1.23 × 10⁵ beads were added to 100 µl of supernatant in 96-well plates and measured as above. Cathepsin D levels were measured by Western blot using the previously published antibody (Journet et al., 1999 (link)).

Starling G.P., Phillips B.A., Ganesh S, & King J.S. (2023). Katnip is needed to maintain microtubule function and lysosomal delivery to autophagosomes and phagosomes. Molecular Biology of the Cell, 34(3), ar12.

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