A21200

Manufactured by Thermo Fisher Scientific

Sourced in United States

The A21200 is a laboratory equipment product offered by Thermo Fisher Scientific. It is a core piece of equipment designed to perform specific functions within a laboratory setting. The description of the A21200's core function is provided in a factual and unbiased manner, without any extrapolation or interpretation of its intended use.

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13 protocols using a21200

Multicolor Imaging of Cytoskeletal Structures

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Cells were permeabilized with 0.25% (PFA-fixed) or 0.1% TX100 (methanol-fixed) and blocked with 5% FBS in standard TBST (0.1% Tween-20 at pH 7.6). To visualize microtubules, methanol-fixed cells were incubated with primary α-tubulin (Sigma-Aldrich T5168, 1:500 dilution) and secondary Alexa Fluor 488-conjugated (Thermo Fisher Scientific A21200, 1:1000 dilution) antibodies for 1 h in blocking solution. TBST was used to wash cells (5 × 5 min). To visualize F-actin, PFA-fixed cells were stained with Alexa Fluor 546 phalloidin (5 units/mL for 20 min in 1% BSA in PBS) and DAPI (2 μM for 20 min in PBS).
Cells were kept in PBS for confocal imaging. Fluorescence and differential interference contrast (DIC) images were collected on an Olympus FV1000 confocal laser-scanning microscope using oil immersion UPLSAPO 60×/1.35 Super Apochromat objective. DAPI, Alexa Fluor 488, Alexa Fluor 546, and SNAP-Cell 647-SiR dyes were excited with 405, 488, 543, and 633 nm lasers, respectively; separate fluorescence signals were collected with 0.5 μm steps in sequential laser mode with emission gates adjusted to avoid spectral overlap.
All sequences and data used in this work are publicly available. Accession IDs for all RefSeq sequences used are provided in Supplemental Tables S1–S4, and IDs for all raw sequencing data are provided in Supplemental Tables S5 and S6.

Benitez-Cantos M.S., Yordanova M.M., O'Connor P.B., Zhdanov A.V., Kovalchuk S.I., Papkovsky D.B., Andreev D.E, & Baranov P.V. (2020). Translation initiation downstream from annotated start codons in human mRNAs coevolves with the Kozak context. Genome Research, 30(7), 974-984.

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ADP Binding to ADAM8 and ADAM33

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For ADP binding to ADAM8, 3000 HEK-A8, HEK-REM, or HEK-EV cells in flow cytometry buffer (1% BSA, 0.1% sodium azide in PBS) were exposed to sera (1:100), supernatants (10 μL), or ADPs (0.1–10 μg/mL). As negative and positive controls, 1 μg/mL normal mouse IgG and goat anti-mouse ADAM8 Ab (AF1031, R&D Systems, RRID: AB_354549), respectively, were analyzed. Alexa Fluor 488 donkey anti-mouse IgG (1:1000, A-21202, Thermo Fisher Scientific, RRID: AB_141607) or Alexa Fluor 488 donkey anti-goat IgG (1:1000, A-11055, Thermo Fisher Scientific, RRID: AB_2534102) were used as secondary Abs. For ADP cross-reactivity to ADAM33, which is primarily cytoplasmic, 10⁶ HEK-A33 or HEK-EV₂ cells were fixed in cold 4% paraformaldehyde and permeabilized with 0.1% Saponin before exposure to ADP or control IgGs (2 μg/50 μL sample). An ADAM33 Ab (LS-C124915, LifeSpan Biosciences, Seattle, WA, USA, RRID: AB_10831502) [2 μg/50 μL sample] and an Alexa Fluor 488 chicken anti-mouse IgG (A-21200, Thermo Fisher Scientific, RRID: AB_2535786) [1.25 μg/50 μL sample] were used as positive control and secondary Ab, respectively. All samples were run on a BD FACSCalibur (BD Biosciences, Woburn, MA, USA).

Mineva N.D., Pianetti S., Das S.G., Srinivasan S., Billiald N.M, & Sonenshein G.E. (2024). A Novel Class of Human ADAM8 Inhibitory Antibodies for Treatment of Triple-Negative Breast Cancer. Pharmaceutics, 16(4), 536.

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Multimodal Imaging of ECM and Cytokines

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Immunofluorescence was performed to assess ECM component and pro-inflammatory cytokine production. After antigen-retrieval, permeabilization, and blocking, multi-color immunofluorescence was produced with 1:200-diluted primary antibodies against an IVD NP-associated notochordal marker (CD24), ECM components (aggrecan and type II collagen), and pro-inflammatory cytokines (TNF-α [NBP2-34372; Novusbio, Centennial, CO, USA] and IL-6 [AF506; R & D Systems, Minneapolis, MN, USA]) for 12 h at 4 °C, followed by incubation with 1:400-diluted Alexa Fluor 488 (A-21200; Thermo Fisher Scientific), 594 (A-21468; Thermo Fisher Scientific), and 647 (A-31573; Thermo Fisher Scientific) secondary antibodies for 1 h at 25 °C. The DAPI was used for counterstaining. Images were captured by fluorescence spectroscopy using the BZ-X700 microscope. The number of positive cells was counted in four random high-power field (×400) using the BZ-X700 software. Immunopositivity was calculated as a percentage relative to DAPI-positive cells (n = 6 per group).

Miyazaki K., Miyazaki S., Yurube T., Takeoka Y., Kanda Y., Zhang Z., Kakiuchi Y., Tsujimoto R., Ohnishi H., Matsuo T., Ryu M., Kuroda R, & Kakutani K. (2022). Protective Effects of Growth Differentiation Factor-6 on the Intervertebral Disc: An In Vitro and In Vivo Study. Cells, 11(7), 1174.

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Multiparameter Flow Cytometry Analysis of LSD1, COVID-19, and ACE2

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Fixed cells were washed with PBS and resuspended in PBS containing 1% BSA prior to antibody staining. Primary antibodies targeting LSD1 (ab17721, Abcam, Cambridge, UK), Coronavirus (FIPV3-70) (sc65653, Santa Cruz Biotechnology, Dallas, TX), and ACE2 (AF594 conjugated, sc390851, Santa Cruz Biotechnology) and secondary chicken anti-mouse AF488 (A21200, Thermo Fisher Scientific, Waltham, MA) and donkey anti-rabbit AF647 (A31573, Thermo Fisher Scientific) antibodies were used. For PBMC samples, CD3 (344838, BioLegend, San Diego, CA), CD4 (558116, BD Biosciences, San Jose, CA), CD8α (555366, BD Biosciences), CD45RA (304133, BioLegend), and CCR7 (353244, BioLegend) antibodies were used. Appropriate antibody controls were used for all flow cytometry stain and acquisition. All samples were acquired on an LSR Fortessa cytometer (BD Biosciences, Franklin Lakes, NJ), and data were analyzed using FlowJo v10 software.

Tu W.J., McCuaig R.D., Melino M., Rawle D.J., Le T.T., Yan K., Suhrbier A., Johnston R.L., Koufariotis L.T., Waddell N., Cross E.M., Tsimbalyuk S., Bain A., Ahern E., Collinson N., Phipps S., Forwood J.K., Seddiki N, & Rao S. (2021). Targeting novel LSD1-dependent ACE2 demethylation domains inhibits SARS-CoV-2 replication. Cell Discovery, 7, 37.

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Establishment of SLAM-expressing Vero Cells

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Vero and 293T cells were cultured in advanced MEM and DMEM media respectively, Media were supplemented with 10% FBS, 1% penicillin/streptomycin, and 1% GlutaMax. Vero cells were transfected either with pCXN2-dolSLAM or pCXN2-phoca-SLAM using standard Lipofectamine 3000 protocols (Thermo Fischer Scientific). At 1 day post-transfection, the cells were cultured in growth media supplemented with 1 mg/ml geneticin G418 (Thermo Fischer Scientific) as a selective antibiotic. Vero-dolSLAM cells and Vero-phocaSLAM cells were sorted into single clones using a MoFlo XDP cell sorter (Beckman Coulter) to ensure an equivalent level of SLAM expression within the cell population. Successfully transfected cells were sorted by staining with anti-HA tag antibody [HA.C5] (1:200; ab18181, Abcam) as the primary antibody and chicken anti-mouse IgG (H + L) Alexa Fluor 488 (1:500; A-21200, Thermo Fischer Scientific) as the secondary antibody. Cell clones were expanded and assessed for SLAM expression using an Attune NxT flow cytometer (Thermo Fischer Scientific).

Jo W.K., Kruppa J., Habierski A., van de Bildt M., Mazzariol S., Di Guardo G., Siebert U., Kuiken T., Jung K., Osterhaus A, & Ludlow M. (2018). Evolutionary evidence for multi-host transmission of cetacean morbillivirus. Emerging Microbes & Infections, 7, 201.

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Riox1 Localization in Transfected HeLa Cells

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HeLa cells were grown to 50–70% confluence on 18 mm diameter coverslips. 24 h post-transfection cells were fixed with 4% paraformaldehyde (10 min). GFP-expressing cells were stained with 1 μg/ml DAPI and slides mounted in Vectashield. For antibody staining cells were permeabilized after fixation with 1% Triton X-100 in PBS and subsequently kept in blocking solution for an hour (10%FCS, 0.2% Tween-20 in PBS). Primary antibodies: α-Riox1 (Mina53, ab169154, Abcam), α-Riox1 (NO66, ab113975, Abcam) and α-UBF (sc-9131, Santa Cruz). Secondary antibodies: Alexa Fluor 488 chicken anti-mouse (A21200, Thermo Fisher), Alexa Fluor 594 donkey anti-rabbit (A21206, Thermo Fisher). Slides were imaged with a fluorescence microscope Carl Zeiss LSM 510 META.

Bräuer K.E., Brockers K., Moneer J., Feuchtinger A., Wollscheid-Lengeling E., Lengeling A, & Wolf A. (2018). Phylogenetic and genomic analyses of the ribosomal oxygenases Riox1 (No66) and Riox2 (Mina53) provide new insights into their evolution. BMC Evolutionary Biology, 18, 96.

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Monocyte Surface Receptor Analysis

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Monocytes were seeded in thermo-sensitive Nunc UpCell 48well plates (VWR, Dietikon, Switzerland) at 5 Â 10 5 cells per well (2 wells/antibody). After treatment, plates were put on ice for 15 min for cell detachment and then distributed in a 96-well plate (Sigma-Aldrich) at 3 Â 10 5 cells per well and incubated 15 min in blocking buffer (1 mM EDTA, 20% HS in PBS) on ice, in the dark. MerTK and CD163 primary antibodies and isotype control (MAB8912, MAB1607 and MAB002, R&D Systems) were added for 1 h, before washing and further incubation with the secondary antibody for 30 min (A21200, Thermo Fisher Scientific). After fixation with 1% PFA, cells were resuspended in 300 mL PBS for immediate analysis. Flow cytometry experiments were read on a CyAn ADM (Beckman Coulter, Nyon, Switzerland) or a FACSCanto II (BD Biosciences). Data analysis was performed with the FlowJo software 7.6.5 (BD Biosciences).

Gauthier A., Fisch A., Seuwen K., Baumgarten B., Ruffner H., Aebi A., Rausch M., Kiessling F., Bartneck M., Weiskirchen R., Tacke F., Storm G., Lammers T, & Ludwig M.G. (2018). Glucocorticoid-loaded liposomes induce a pro-resolution phenotype in human primary macrophages to support chronic wound healing. Biomaterials, 178.

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Fluorescent Antibody Labeling Protocol

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Alexa Fluor 488 chicken anti-mouse immunoglobulin G (IgG) (Invitrogen #A21200, RRID_AB_2535786, used at 1:400); Alexa Fluor 488 chicken anti-rabbit IgG (Invitrogen #A21441, RRID_AB_10563745, used at 1:400); Alexa Fluor 568 donkey anti-sheep IgG (Invitrogen #A21099, RRID_AB_10055702, used at 1:400); Alexa Fluor Plus 488 goat anti-rabbit (ThermoFisher A32731, used at 1:500); Alexa Fluor Plus 594 goat anti-mouse (ThermoFisher A32742, used at 1:500); and goat anti-mouse IgG-HRP (horseradish peroxidase) (BioRad #170-6516, RRID:AB_11125547, used at 1:20000).

Gordon D.E., Hiatt J., Bouhaddou M., Rezelj V.V., Ulferts S., Braberg H., Jureka A.S., Obernier K., Guo J.Z., Batra J., Kaake R.M., Weckstein A.R., Owens T.W., Gupta M., Pourmal S., Titus E.W., Cakir M., Soucheray M., McGregor M., Cakir Z., Jang G., O’Meara M.J., Tummino T.A., Zhang Z., Foussard H., Rojc A., Zhou Y., Kuchenov D., Hüttenhain R., Xu J., Eckhardt M., Swaney D.L., Fabius J.M., Ummadi M., Tutuncuoglu B., Rathore U., Modak M., Haas P., Haas K.M., Naing Z.Z., Pulido E.H., Shi Y., Barrio-Hernandez I., Memon D., Petsalaki E., Dunham A., Marrero M.C., Burke D., Koh C., Vallet T., Silvas J.A., Azumaya C.M., Billesbølle C., Brilot A.F., Campbell M.G., Diallo A., Dickinson M.S., Diwanji D., Herrera N., Hoppe N., Kratochvil H.T., Liu Y., Merz G.E., Moritz M., Nguyen H.C., Nowotny C., Puchades C., Rizo A.N., Schulze-Gahmen U., Smith A.M., Sun M., Young I.D., Zhao J., Asarnow D., Biel J., Bowen A., Braxton J.R., Chen J., Chio C.M., Chio U.S., Deshpande I., Doan L., Faust B., Flores S., Jin M., Kim K., Lam V.L., Li F., Li J., Li Y.L., Li Y., Liu X., Lo M., Lopez K.E., Melo A.A., Moss FR I.I.I., Nguyen P., Paulino J., Pawar K.I., Peters J.K., Pospiech TH J.r., Safari M., Sangwan S., Schaefer K., Thomas P.V., Thwin A.C., Trenker R., Tse E., Tsui T.K., Wang F., Whitis N., Yu Z., Zhang K., Zhang Y., Zhou F., Saltzberg D., Hodder A.J., Shun-Shion A.S., Williams D.M., White K.M., Rosales R., Kehrer T., Miorin L., Moreno E., Patel A.H., Rihn S., Khalid M.M., Vallejo-Gracia A., Fozouni P., Simoneau C.R., Roth T.L., Wu D., Karim M.A., Ghoussaini M., Dunham I., Berardi F., Weigang S., Chazal M., Park J., Logue J., McGrath M., Weston S., Haupt R., Hastie C.J., Elliott M., Brown F., Burness K.A., Reid E., Dorward M., Johnson C., Wilkinson S.G., Geyer A., Giesel D.M., Baillie C., Raggett S., Leech H., Toth R., Goodman N., Keough K.C., Lind A.L., Klesh R.J., Hemphill K.R., Carlson-Stevermer J., Oki J., Holden K., Maures T., Pollard K.S., Sali A., Agard D.A., Cheng Y., Fraser J.S., Frost A., Jura N., Kortemme T., Manglik A., Southworth D.R., Stroud R.M., Alessi D.R., Davies P., Frieman M.B., Ideker T., Abate C., Jouvenet N., Kochs G., Shoichet B., Ott M., Palmarini M., Shokat K.M., García-Sastre A., Rassen J.A., Grosse R., Rosenberg O.S., Verba K.A., Basler C.F., Vignuzzi M., Peden A.A., Beltrao P, & Krogan N.J. (2020). Comparative host-coronavirus protein interaction networks reveal pan-viral disease mechanisms. Science (New York, N.y.), 370(6521), eabe9403.

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Immunohistochemistry of Drosophila Larval and Adult Brains

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Larval and adult brains were dissected and fixed in 4% formaldehyde in PBS with 0.2% Triton X-100 for 20 min at room temperature. For larval brains, samples were incubated with primary antibodies at 4°C overnight and secondary antibodies at room temperature for 2 h. For adult brains, samples were incubated with primary antibodies at 4°C for 2 nights and secondary antibodies at room temperature for 2 h. The following primary antibodies were used at the indicated dilutions: rat anti-Elav [Developmental Studies Hybridoma Bank (DSHB), 7E8A10; 1:50], mouse anti-Prospero (DSHB, MR1A; 1:10), mouse nc-82 (DSHB; 1:20), rabbit anti-phosphohistone H3 (Cell Signaling, 9701S; 1:200), chicken anti-GFP (Abcam, 13970; 1:2000), rabbit anti-Ase (Brand et al., 1993 (link)) (1:200), guinea pig anti-Deadpan (provided by James Skeath, Department of Genetics, Washington University of St. Louis; 1:2000) and anti-Eyeless (Kammermeier et al., 2001 (link)) (1:200). Secondary antibodies were Alexa Fluor 405-, Alexa Fluor 555-, Alexa Fluor 633-, or Alexa Fluor 488-conjugated (Invitrogen, A-11039, A-21435, A-21105, A-21072, A-21094, A-21200) and used at 1:300, 1:500, 1:300 and 1:500, respectively. DNA stain was DAPI (Sigma). Samples were mounted in Vectashield. Quantification was performed using ImageJ and Imaris (Bitplane).

Weng R, & Cohen S.M. (2015). Control of Drosophila Type I and Type II central brain neuroblast proliferation by bantam microRNA. Development (Cambridge, England), 142(21), 3713-3720.

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Double Immunofluorescence Staining of pHSCs

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For double immunofluorescence staining, cultured human pHSCs on coverslips or cytospun mouse pHSCs were fixed and permeabilized with 4% paraformaldehyde and Triton X-100, respectively. These cells were washed with TBS and blocked in protein blocking solution (X9090; Dako) for 30 min and incubated with first primary antibody, CD44ICD (KAL-KO601; CosmoBio; Carlsbad, CA, USA) or MMP14 (af918; R&D Systems) at 4 °C overnight and followed by Alexa Fluor 568-conjugated goat anti-rabbit IgG (A10042, Invitrogen) or Alexa Fluor 647-conjugated donkey anti-goat IgG (A21447, Invitrogen) for 30 min. For second primary antibodies, sections were incubated with α-SMA (A5228; Sigma-Aldrich) or CD44 (ab157107; Abcam) for 2 h at room temperature and followed by Alexa Fluor 488-conjugated chicken anti-mouse IgG (A21200, Invitrogen) or Alexa Fluor 568-conjugated goat anti-rabbit IgG (A10042, Invitrogen) for 30 min. 4’,6-diamidino-2-phenylindole (DAPI) were employed in the counterstaining procedure. The specimens were then observed and analyzed by confocal microscope DMi8-S (Leica Microsystems, Wetzlar, Germany) or CLSM21 (Carl Zeiss Inc., Thornwood, NY, USA).

Han J., Lee C., Jeong H., Jeon S., Lee M., Lee H., Choi Y.H, & Jung Y. (2024). Tumor necrosis factor-inducible gene 6 protein and its derived peptide ameliorate liver fibrosis by repressing CD44 activation in mice with alcohol-related liver disease. Journal of Biomedical Science, 31, 54.

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