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The D9542 is a laboratory centrifuge designed for general-purpose applications. It features a compact, durable construction and operates at a maximum speed of 6,000 rpm. The centrifuge is suitable for a variety of sample types and volumes, making it a versatile tool for researchers and laboratory technicians.

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Lab products found in correlation

Draq5, by Thermo Fisher Scientific (3 mentions) Alexa fluor 488 streptavidincomplex, by Jackson ImmunoResearch (2 mentions) A1r point scanninglaser confocal microscope, by Nikon (2 mentions) Neurotrace 640, by Thermo Fisher Scientific (2 mentions) Prolong diamond antifade mountant, by Thermo Fisher Scientific (2 mentions) P8920, by Thermo Fisher Scientific (2 mentions) Prolong gold antifade mounting media, by Thermo Fisher Scientific (2 mentions) A7030, by Merck Group (2 mentions) Ab30682, by Abcam (1 mentions) A1r hd25, by Nikon (1 mentions) Cy3 conjugated goat anti rabbit igg, by Abcam (1 mentions) Fitc conjugated goat anti mouse igg, by Abcam (1 mentions) Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions) Paraformaldehyde (pfa), by Merck Group (1 mentions) Triton x 100, by Merck Group (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Alexa fluor 647 phalloidin, by Thermo Fisher Scientific (1 mentions) A12381, by Thermo Fisher Scientific (1 mentions) Alexa fluor 594 phalloidin, by Thermo Fisher Scientific (1 mentions) Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)

11 protocols using d9542

1

Immunofluorescence Staining of HTNV NP and SREBP2

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Cells exposed to the indicated treatment were fixed with ice-cold 4% (w/v) paraformaldehyde (PFA, Sigma–Aldrich) for 15 min and then permeabilized with 0.1% Triton X-100 (Sigma–Aldrich) for 20 min at RT. After blocking with 3% bovine serum albumin (BSA, Sigma–Aldrich) for 30 min, specific primary antibodies, namely, anti-HTNV NP 1A8 mouse monoclonal antibody (prepared and maintained by our laboratory; 1:50 dilution) and anti-SREBP2 rabbit polyclonal antibody (Abcam, ab30682; 1:50 dilution), were added and incubated overnight at 4°C. After five washes with DPBS, the secondary antibodies, namely, FITC-conjugated goat anti-mouse IgG (Abcam, ab6785) and Cy3-conjugated goat anti-rabbit IgG (Abcam, ab6939), were used for detection (incubation at 37°C for 1 h). Cell nuclei were stained with DAPI (Thermo Fisher, D9542) for 5 min at RT. After sealing with ProLong™ Gold Antifade Mountant (Thermo Fisher, P36930), the samples were observed using a fluorescence microscope (A1R-HD25, Nikon).
Yang Y., Li M., Ma Y., Ye W., Si Y., Zheng X., Liu H., Cheng L., Zhang L., Zhang H., Zhang X., Lei Y., Shen L., Zhang F, & Ma H. (2022). LncRNA NEAT1 Potentiates SREBP2 Activity to Promote Inflammatory Macrophage Activation and Limit Hantaan Virus Propagation. Frontiers in Microbiology, 13, 849020.
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2

Mosquito Stylet Immunostaining Protocol

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7 to14 day-old mosquitoes were anesthetized on ice. Stylets were dissected and placed directly into a 24 well-plate containing 4% paraformaldehyde, 0.1 M Millonig’s Phosphate Buffer (pH 7.4) and 0.25% Triton X-100. All subsequent steps were performed on a low-speed orbital shaker at room temperature. Samples were washed at least 4 times 15 min in PTx.2 (for 1L: 100 mL PBS 10x, 2 mL TritonX-100) before placed overnight in iDISCO permeabilization solution (for 500 mL: 400 mL PTx.2, 11.5 g glycine, 100 mL DMSO) (Renier et al., 2014 (link)). Samples were then incubated in iDISCO PTwH solution (for 1L: 100 mL 10x PBS, 2mL Tween-20, 1 mL of 10mg/mL Heparin stock solution) with 5% DMSO for at least 2 days at room temperature with the following reagents: (1) 1:20 AlexaFluor 594 phalloidin (ThermoFisher #A12381) (Figure 3F) or (2) 1:20 AlexaFluor 488 phalloidin (ThermoFisher #A12379) and 1:500 DAPI (Millipore Sigma #D9542) (Figure S2D) or (3) 1:20 AlexaFluor 647 phalloidin (ThermoFisher #A22287) (Figure S2G) or (4) 2 mg/mL FITC (Millipore Sigma #1.24546) (Figure S2A). Samples were then washed at least 4 times 15 min with PTx.2 solution and mounted in Vectashield. If a sample contained AlexaFluor 647, it was mounted in SlowFade instead of Vectashield because this fluorophore was better preserved in this mounting medium.
Jové V., Gong Z., Hol F.J., Zhao Z., Sorrells T.R., Carroll T.S., Prakash M., McBride C.S, & Vosshall L.B. (2020). Sensory Discrimination of Blood and Floral Nectar by Aedes aegypti Mosquitoes. Neuron, 108(6), 1163-1180.e12.
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3

Neuroanatomical Labeling and Microscopy Protocol

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After recording, fish were deeply anesthetized with a concentrated
solution of MS:222 (1:10,000) and perfused through the heart with a teleost
Ringer solution followed by a fixative, consisting of 4% paraformaldehyde in
0.1 M phosphate buffer. The brains were postfixed for 12–24 h,
cryoprotected with 30% sucrose, and sectioned at 60 mm on a cryostat.
Sections were subsequently processed with an Alexa Fluor 488 Streptavidin
complex (Jackson Immuno Research Laboratories; Antibody ID - AB_2337249; at
1:500) to label the biocytin filled cells, and DAPI (Sigma Aldrich # D9542;
at 1:1000) and NeuroTrace 640 (ThermoFisher Scientific #N21483; at 1:500) to
visualize the layers of ELL. Sections were then mounted on slides, dried of
excess PBS, and coverslipped with either VectaShield Antifade (Vector
Laboratories # H-1000-10) or Molecular Probes™ ProLong™
Diamond Antifade Mountant (Fisher Scientific; Molecular Probes™
P36965). Morphologically recovered neurons were inspected and subsequently
photographed using a confocal microscope (Inverted Nikon A1R point-scanning
laser confocal microscope with high-sensitivity GaAsP detectors) with either
a 20× air objective or a 40× oil immersion objective. Images
were collapsed along the Z-dimension implementing the maximum brightness per
pixel. Each fluorescence channel was pseudo-colored as specified in Figure 2.
Perks K.E, & Sawtell N.B. (2022). Neural readout of a latency code in the active electrosensory system. Cell reports, 38(13), 110605.
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4

Neuroanatomical Labeling and Microscopy Protocol

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After recording, fish were deeply anesthetized with a concentrated
solution of MS:222 (1:10,000) and perfused through the heart with a teleost
Ringer solution followed by a fixative, consisting of 4% paraformaldehyde in
0.1 M phosphate buffer. The brains were postfixed for 12–24 h,
cryoprotected with 30% sucrose, and sectioned at 60 mm on a cryostat.
Sections were subsequently processed with an Alexa Fluor 488 Streptavidin
complex (Jackson Immuno Research Laboratories; Antibody ID - AB_2337249; at
1:500) to label the biocytin filled cells, and DAPI (Sigma Aldrich # D9542;
at 1:1000) and NeuroTrace 640 (ThermoFisher Scientific #N21483; at 1:500) to
visualize the layers of ELL. Sections were then mounted on slides, dried of
excess PBS, and coverslipped with either VectaShield Antifade (Vector
Laboratories # H-1000-10) or Molecular Probes™ ProLong™
Diamond Antifade Mountant (Fisher Scientific; Molecular Probes™
P36965). Morphologically recovered neurons were inspected and subsequently
photographed using a confocal microscope (Inverted Nikon A1R point-scanning
laser confocal microscope with high-sensitivity GaAsP detectors) with either
a 20× air objective or a 40× oil immersion objective. Images
were collapsed along the Z-dimension implementing the maximum brightness per
pixel. Each fluorescence channel was pseudo-colored as specified in Figure 2.
Perks K.E, & Sawtell N.B. (2022). Neural readout of a latency code in the active electrosensory system. Cell reports, 38(13), 110605.
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5

Measuring Cell Invasion Using Matrigel Assay

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To measure invasion, a two-chamber assay was performed. To this end, 40,000 cells were seeded in 50% Matrigel in collagen-coated inserts for 24-well plates (BD, 353097, 8 µm pore size). Serum-free medium ± TGFβ was added to the chamber, while fetal bovine serum-containing growth medium was provided in the wells. Matrigel was removed after 48 h incubation, cells were fixed by 20 min 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) at room temperature (RT) prior to 4,6-diamidino-2-phenylindole (DAPI) staining (Sigma-Aldrich, D9542; 1:2000) and mounting (Thermo Scientific, 9990402).
Hasselluhn M.C., Schmidt G.E., Ellenrieder V., Johnsen S.A, & Hessmann E. (2019). Aberrant NFATc1 signaling counteracts TGFβ-mediated growth arrest and apoptosis induction in pancreatic cancer progression. Cell Death & Disease, 10(6), 446.
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6

Single-Cell RNA-Seq of Rhabdomyosarcoma Tumoroids

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For each tumoroid model (RMS127 and RMS444), viably frozen cells were thawed and resuspended in BM1* medium. Prior to sorting, 4′,6‐diamidino‐2‐phenylindole (DAPI, Sigma‐Aldrich, #D9542) and DRAQ5 (Thermo Fisher, #65‐0880‐92) were added to the single‐cell suspensions to achieve final concentrations of 1 and 5 μM, respectively. Viable single‐cells (DAPI−, DRAQ5+, FSC/SSC) were then sorted into 384‐well plates (BioRad, #HSP3801) containing 10 μl of mineral oil (Sigma, #M5310) and 50 nl of barcoded RT primers using a SONY SH800S Cell Sorter. Libraries were prepared according to the SORT‐seq protocol (Muraro et al, 2016 (link)) and sequenced on the Illumina NextSeq500 (paired‐end, 75 bp).
Meister M.T., Groot Koerkamp M.J., de Souza T., Breunis W.B., Frazer‐Mendelewska E., Brok M., DeMartino J., Manders F., Calandrini C., Kerstens H.H., Janse A., Dolman M.E., Eising S., Langenberg K.P., van Tuil M., Knops R.R., van Scheltinga S.T., Hiemcke‐Jiwa L.S., Flucke U., Merks J.H., van Noesel M.M., Tops B.B., Hehir‐Kwa J.Y., Kemmeren P., Molenaar J.J., van de Wetering M., van Boxtel R., Drost J, & Holstege F.C. (2022). Mesenchymal tumor organoid models recapitulate rhabdomyosarcoma subtypes. EMBO Molecular Medicine, 14(10), e16001.
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7

Quantification of Nucleic Acids in SEC Fractions

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DNA or RNA was detected by incubating samples from SEC fractions in black 96-well plates with DAPI at a concentration of 0.12 μg/mL (Sigma-Aldrich, catalog no. D9542) or SYBR Gold at a 1/10000 dilution (Invitrogen, catalog no. S11494) in 100 mM NaCl, 20 mM HEPES (pH 7.9), and 10 mM EDTA. Fluorescence was measured with a BMG PolarSTAR Omega plate reader using a λex of 350 nm and a λem of 475 nm for DAPI and a λex of 485 nm and a λem of 535 nm for SYBR Gold. Proteins were digested with 20 μg of proteinase K (Amresco, catalog no. 0706) at 37 °C for 1 h, and nucleic acids with DNase I (Worthington Biochemical, catalog no. LS006353) or RNase A (Sigma-Aldrich, catalog no. R4642) at room temperature for 1 h.
Thompson V.F., Victor R.A., Morera A.A., Moinpour M., Liu M.N., Kisiel C.C., Pickrel K., Springhower C.E, & Schwartz J.C. (2018). Transcription-Dependent Formation of Nuclear Granules Containing FUS and RNA Pol II. Biochemistry, 57(51), 7021-7032.
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8

Imaging Cells After Isoproterenol Treatment

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Cells were plated on poly-D-lysine (Sigma, P8920) coated coverslips (Fisher, 12–545-100) in 12 well plates. Cells untreated or treated with 100 nM Iso for 45 minutes at 37°C. Cells fixed in 3.7% formaldehyde (Fisher, F79) in modified BRB80 (80 mM PIPES pH 6.8, 1 mM MgCl2, 1 mM CaCl2) for 20 minutes. For cells to be stained, cells were blocked in 3% BSA (Sigma, A7030), 0.1% Triton X-100 (Sigma, T8787) in PBS. Next, cells were stained using antibodies (Supplementary Table 6) and with 1 µg/ml DAPI (20 mg/ml stock in water, Sigma, D9542) or 1 μM DRAQ5 (Invitrogen, 62251) in PBS. Then the coverslips were mounted on glass slides with ProLong Gold Antifade mounting media (Invitrogen, P10144). Slides were stored for a minimum of 24 hours to allow mounting media to dry before imaging.
Peng G.E., Pessino V., Huang B, & von Zastrow M. (2021). Spatial decoding of endosomal cAMP signals by a metastable cytoplasmic PKA network. Nature chemical biology, 17(5), 558-566.
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9

Immunodetection of Calcium Channels and Serotonin Receptors

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Voltage-gated calcium channels were detected with rabbit polyclonal antibodies from Alomone (CaV3.1:ACC-021 and CaV3.3:ACC-009) in a concentration 1:50. 5-HT was detected with a rat monoclonal antibody (1:100; Milipore). 5-HT2C receptors were detected by a mouse monoclonal IgG1 antibody (BD Pharmigen; clone SR-2C) in concentration 1:100. Microtubule associated protein 2 (MAP2) was detected with a mouse monoclonal IgG1 antibody (Sigma; clone HM-2) in concentration 1:300 or rabbit polyclonal IgG (Santa-Cruz, MAP-2 Antibody (H-300) in concentration 1:100), DAPI nucleic acid stain (1 mg/ml; Invitrogen D-9542) in concentration 1:300.
Petersen A.V., Jensen C.S., Crépel V., Falkerslev M, & Perrier J.F. (2017). Serotonin Regulates the Firing of Principal Cells of the Subiculum by Inhibiting a T-type Ca2+ Current. Frontiers in Cellular Neuroscience, 11, 60.
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10

Imaging Cells After Isoproterenol Treatment

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Cells were plated on poly-D-lysine (Sigma, P8920) coated coverslips (Fisher, 12–545-100) in 12 well plates. Cells untreated or treated with 100 nM Iso for 45 minutes at 37°C. Cells fixed in 3.7% formaldehyde (Fisher, F79) in modified BRB80 (80 mM PIPES pH 6.8, 1 mM MgCl2, 1 mM CaCl2) for 20 minutes. For cells to be stained, cells were blocked in 3% BSA (Sigma, A7030), 0.1% Triton X-100 (Sigma, T8787) in PBS. Next, cells were stained using antibodies (Supplementary Table 6) and with 1 µg/ml DAPI (20 mg/ml stock in water, Sigma, D9542) or 1 μM DRAQ5 (Invitrogen, 62251) in PBS. Then the coverslips were mounted on glass slides with ProLong Gold Antifade mounting media (Invitrogen, P10144). Slides were stored for a minimum of 24 hours to allow mounting media to dry before imaging.
Peng G.E., Pessino V., Huang B, & von Zastrow M. (2021). Spatial decoding of endosomal cAMP signals by a metastable cytoplasmic PKA network. Nature chemical biology, 17(5), 558-566.
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