Hulamixer

Manufactured by Thermo Fisher Scientific

Sourced in United States, Norway

The HulaMixer is a laboratory mixing platform designed for gentle, end-over-end mixing of samples. It provides a consistent and reliable mixing motion to facilitate various laboratory workflows.

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Lab products found in correlation

29 protocols using hulamixer

Filipin Staining of Cholesterol

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Filipin III (Santa Cruz Biotechnology) was dissolved in DMSO at 5 mg/mL, aliquoted and stored under N₂ at − 80 °C until use. CU428 and II8G-IA cultures at ~ 3 × 10⁵ cells/mL were treated with cholesterol in the presence or absence of U18666A as indicated above. After 1 h of incubation, samples of 200 µL were transferred to microcentrifuge tubes, centrifuged at 1000 × g, washed with 1 mL of 10 mM Tris–HCl pH 7.5 and resuspended in 100 µL Tris–HCl. Cells were fixed through the addition of an equal volume of 4% paraformaldehyde—3.4% sucrose in phosphate buffer saline (PBS) for 15 min at room temperature. They were then washed twice with 400 µL PBS and resuspended in 100 µL PBS. Staining was performed with 50 µg/mL Filipin for 1 h at room temperature with rotation in a HulaMixer (Invitrogen). Finally, the cells were washed twice with 400 µL PBS and resuspended in 40 µL PBS.
Cells mounted under coverslips were observed and photographed using a Nikon Eclipse E-800 epifluorescence microscope equipped with an Andor Clara DR-1306 monochrome camera. Filipin staining was visualized using a UV-2A filter cube. After being taken, the images were processed and analyzed using the Fiji software^{44 (link)}.

Hernández J., Gabrielli M., Costa J, & Uttaro A.D. (2021). Phagocytic and pinocytic uptake of cholesterol in Tetrahymena thermophila impact differently on gene regulation for sterol homeostasis. Scientific Reports, 11, 9067.

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Magnetic Bead-based FLAG-tag Isolation of Stable HEK293T Cells

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In order to select the more efficient magnetic beads for isolating the stable HEK293T cells via 3x FLAG®-tag (DYKDDDK) peptide (F4799, Sigma) (Flag tag), MACS was performed by using two different magnetic beads (Fig. 2); anti-flag M2 magnetic beads (Sigma, M8823) are 4% agarose beads already bound with anti-Flag M2 (mouse monoclonal antibody) and Flag antibody (R&D, IC8529V, monoclonal rabbit) pre-coated Dynabeads (Invitrogen, 11203D, sheep anti-rabbit IgG) are 2.8 μm superparamagnetic beads magnetic beads with affinity-purified polyclonal sheep anti-rabbit IgG covalently bound to the bead surface. Transduced HEK293T cells were incubated with magnetic beads at room temperature over 1 h on a HulaMixer (Invitrogen, 15920D) and then the cell-bound magnetic beads were isolated by using a magnetic rack, DynaMag-2 Magnet (Invitrogen).

Rufino-Ramos D., Lule S., Mahjoum S., Ughetto S., Bragg D.C., de Almeida L.P., Breakefield X.O, & Breyne K. (2022). Using genetically modified extracellular vesicles as a non-invasive strategy to evaluate brain-specific cargo. Biomaterials, 281, 121366.

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Whole Blood Assay for Meningococcal Cytokine Response

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After signed consent was obtained, whole blood from healthy volunteers was drawn
into 3.5 ml citrate tubes (Vacuette 3.5 ml; Greiner Bio One Gmbh, Kremsmünster,
Austria), and the first tube was discarded. Whole blood was immediately
incubated with heat-inactivated N. meningitidis wt, N.
meningitidis lpxL1 mutant (both 10⁷/ml) or vehicle (TBS)
[37℃, 12 rpm Hulamixer (Invitrogen Dynal AS, Oslo Norway)].
To determine the optimal time point to measure pro-inflammatory cytokines, whole
blood from four donors was centrifuged [2500 g, 15 min at room
temperature (RT)] after 0, 4, 6 and 12 h, and plasma was collected for
measurements of cytokines.
After these initial experiments, an incubation period of 4 h was chosen, and
whole blood from six donors was incubated with increasing concentrations of
N. meningitidis wt or N. meningitidis
lpxL1 mutant (10⁶/ml, 10⁷/ml or
10⁸/ml). In some cases, rhIL-10 (25 ng/ml) or vehicle (TBS) was
added to whole blood 15 min prior to the addition of the bacteria. Similar IL-10
concentrations have previously been used in in vitro studies,^{29 (link)} and have also been observed in patients with severe fulminant
meningococcal sepsis.^{30 (link)}

Aass H.C., Hellum M., Trøseid A.M., Brandtzaeg P., Berg J.P., Øvstebø R, & Henriksson C.E. (2018). Whole-blood incubation with the Neisseria meningitidis lpxL1 mutant induces less pro-inflammatory cytokines than the wild type, and IL-10 reduces the MyD88-dependent cytokines. Innate Immunity, 24(2), 101-111.

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Antibody-based ProGRP Immunoaffinity Extraction

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To remove any unbound anti-ProGRP the antibody-coated magnetic beads were prewashed as described elsewhere:^{14 (link)} The desired volume of beads was washed with 1 mL PBS containing 0.05% Tween 20, and re-dissolved in PBS, yielding a solution with the initial bead concentration, ready for use.
The immunoaffinity extraction was performed as follows using magnetic beads: Protein LoBind Eppendorf tubes containing the sample (an in-solution digest of the protein standard or a protein precipitated, diluted and digested serum sample) were added 20 μL of prewashed antibody-coated magnetic beads. To capture the peptides the Eppendorf tubes were rotated and shaken for 1 h on a HulaMixer (Invitrogen), to facilitate the epitope–antibody interaction. The Eppendorf tubes were then placed in the magnetic rack (DynaMag-2 from Invitrogen) to collect the beads and remove the solution. The beads were then washed with 500 μL of PBS containing 0.05% Tween 20, 500 μL of PBS, 300 μL of Tris–HCl (pH 7.4), and 300 μL of 50 mM ABC solution, prior to elution.

Levernæs M.C., Farhat B., Oulie I., Abdullah S.S., Paus E., Reubsaet L, & Halvorsen T.G. (2019). Immunocapture sample clean-up in determination of low abundant protein biomarkers – a feasibility study of peptide capture by anti-protein antibodies. RSC Advances, 9(60), 34902-34911.

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Exosomal Protein Isolation and Detection

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Ten μl of Exosome – Human CD63 Isolation/Detection (Invitrogen) or Exosome – Human CD9 (Invitrogen) were added to samples and incubated on a HulaMixer™ sample mixer at 4°C overnight. The following day the beads were briefly centrifuged, separated by a magnet from the supernatant, washed and resuspended in PBS.

O’Brien K., Ughetto S., Mahjoum S., Nair A.V, & Breakefield X.O. (2022). Uptake, functionality, and re-release of extracellular vesicle-encapsulated cargo. Cell reports, 39(2), 110651.

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Immunoprecipitation and Transient Transfection

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For immunoprecipitation experiment, cells were lysed by ice-cold TX-100 lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM DTT, 1% Triton X-100 [MP bio, 9002-93-1]) supplemented with protease and phosphatase inhibitors. Gently sonication was used to completely lyse cells and shear DNA. For co-immunoprecipitation, cells were prepared in pre-cold Pierce™ IP buffer (Thermo Fisher Scientific, 87787) supplemented with protease inhibitors. The cell lysate was gently rotated at 4°C for 4 h (HulaMixer, Invitrogen) and then centrifuged at 16,000 g for 15 min at 4°C. The indicated antibodies were incubated with the supernatants of cell lysates and gently rotated overnight at 4°C, subsequently mixed with pre-washed protein A/G-plus agarose beads (Santa Cruz Biotechnology, sc-2003) at 4°C for 4 h. Immunoprecipitates were isolation by centrifuging samples at 1,000 g for 3 min and washed 5 times with the lysis buffer before being separated by SDS-PAGE and immunoblotted with indicated antibodies.
For transient transfection experiments, HEK293T and HeLa cells were seeded in culture dishes and incubated for approximately 24 h. Cells were then transfected with indicated pcDNA3.1-based expression vectors by Lipofectamine 2000 transfection reagent for another 24 h.

Xia L., Nie T., Lu F., Huang L., Shi X., Ren D., Lu J., Li X., Xu T., Cui B., Wang Q., Gao G, & Yang Q. (2023). Direct regulation of FNIP1 and FNIP2 by MEF2 sustains MTORC1 activation and tumor progression in pancreatic cancer. Autophagy, 20(3), 505-524.

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Fluorescent Labeling of Erythrocytes

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Erythrocytes (500 μL, 3 x 10⁸ cells/mL) were added to fluorescein isothiocyanate, carboxyfluorescein-SE or TAMRA-SE in Dulbecco’s phosphate buffered saline solution (1 mg/mL, 500 μL, pH 8.0) and delicately inverted (2 hrs, 1 rpm, HulaMixer™ (Invitrogen), 4 °C). Erythrocytes were washed with Dulbecco’s solution (15x times, 10 mL) and collected with centrifugation (800 rpm, 8 minutes). Labelling was confirmed with fluorescence microscopy using a Nikon Eclipse TE300 equipped with a Plan Fluor 40x 0.75 NA objective and CoolLED pE-4000 and pE-100 light source. Labelled erythrocytes were suspended in Alsever’s solution and stored in a light protected container at 4 °C.

Chauhan V.M, & Pritchard D.I. (2018). Haematophagic Caenorhabditis elegans. Parasitology, 146(3), 314-320.

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Carboplatin Loading on Nanosomes

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A total of 100 mg of nanosomes were accurately weighted in Eppendorf tubes and a 2 mg/mL CBP solution prepared in 0.1 M phosphate-buffered saline (PBS) pH 5 was put in contact with the nanosomes for 24 h at room temperature. The suspensions were placed on a HulaMixer (Invitrogen) under continuous shaking, using the following parameters: orbital shaking 3 rpm (90 s), reciprocal shaking 1° (10 s), and vibration movement 1° (10 s). After 24 h, the suspensions were centrifuged for 10 min at 12,000 rpm and 350 μL of the supernatant was sampled and tested using either electrochemical or spectrophotometric methods. For the electrochemical method, a 1:1 dilution with 0.1 M PBS pH 5 was performed before testing, while for the spectrophotometric method no dilution was required. The CBP concentration was determined using the corresponding calibration curves and the encapsulation efficiency (EE%) and loading capacity (LC%) were calculated using the following equations [32 (link)]:

where V—volume of the release media, C_i—initial CBP concentration, C_f—final CBP concentration (after loading), and m_{MCPs-carboplatin}—weight of the loaded nanosomes.

Pusta A., Tertis M., Ardusadan C., Mirel S, & Cristea C. (2024). Electrochemical Sensing Device for Carboplatin Monitoring in Proof-of-Concept Drug Delivery Nanosystems. Nanomaterials, 14(9), 793.

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Interaction of JEV RNA with Host Proteins

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Interaction of JEV RNA with GRP78, PHB and hnRNPC was analysed by the method described earlier.^{42 (link)} Briefly, non-infected or infected hNS1 cells were treated with 1% formaldehyde for 10 min at RT in Hula mixer (Invitrogen) followed by addition of 0.25 M glycine of pH 7.0. These cross-linked cells were sonicated to get the protein complex. Precleared lysate was obtained by addition of dynabeads containing protein G (Novex, Life Technologies, USA). The beads were coated with respective antibodies and allowed to rotate alongwith precleared supernatant at RT for 90 min. The beads were washed thoroughly with antibody binding and washing buffer supplied in the immunoprecipitation kit. The complexes were eluted by the elution buffer. RNA was extracted from the elute samples by conventional method using Trizol reagent (Sigma). RNAs obtained were used in RT-PCR with JEV primers and reaction without reverse transcriptase was used as control. The PCR products were analysed in 2% agarose gel. Purified PCR products (PCR Purification Kit, Qiagen) of JE-positive samples were sent for sequencing. Sequencing was done by Invitrogen Bioservices India Pvt. Ltd.

Mukherjee S., Singh N., Sengupta N., Fatima M., Seth P., Mahadevan A., Shankar S.K., Bhattacharyya A, & Basu A. (2017). Japanese encephalitis virus induces human neural stem/progenitor cell death by elevating GRP78, PHB and hnRNPC through ER stress. Cell Death & Disease, 8(1), e2556-.

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Immunoprecipitation and Transient Transfection

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