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Fjk 16s

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The FJK-16s is a laboratory equipment designed for centrifugation. It is capable of separating different components of a liquid sample based on their densities. The device features a maximum speed of 16,000 revolutions per minute and can accommodate various rotor types to accommodate different sample volumes and tube sizes.

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Lab products found in correlation

Facsdiva software, by BD (4 mentions) Foxp3 staining buffer set, by Thermo Fisher Scientific (3 mentions) Ionomycin, by Merck Group (3 mentions) Anti cxcr5 2g8, by BD (2 mentions) 220 ra3 6b2, by BD (2 mentions) Rm4 5, by Thermo Fisher Scientific (2 mentions) Golgiplug, by BD (2 mentions) Cd25 7d4, by Thermo Fisher Scientific (2 mentions) Live dead fixable dead cell stain kit, by Thermo Fisher Scientific (2 mentions) Lsm 700, by Zeiss (2 mentions) Cd103 m290, by BD (1 mentions) Cd8 53 6, by BD (1 mentions) Cytofix cytoperm kit, by BD (1 mentions) Kaluza software, by Beckman Coulter (1 mentions) Gallios flow cytometer, by Beckman Coulter (1 mentions) Rabbit anti mouse insulin antibodies, by Cell Signaling Technology (1 mentions) Donkey anti rabbitalexafluor647 antibodies, by Dianova (1 mentions) Dylight 488, by Dianova (1 mentions) Biotinylated horse anti rabbit antibody, by Vector Laboratories (1 mentions) Uc10 4b9, by Thermo Fisher Scientific (1 mentions)

15 protocols using fjk 16s

1

Multiparametric Flow Cytometry Immunophenotyping

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Fluorescence dye labeled Abs specific for CD4 (L3T4), B220 (RA3-6B2), CD44 (IM7), Fas (15A7), IgM (II/41), T- and B-cell activation antigen (GL7), ICOS (C398.4A), PD-1 (J43), IFNγ (XMG1.2), IL-4 (11B11), IL-17A (eBio17B7), IL-21 (FFA21), Bcl-6 (K112-91) and FoxP3 (FJK-16s) were purchased from BD, eBioscience and Biolegend. Analysis of CXCR5 expression was performed using a biotinylated anti-CXCR5 (2G8, BD) antibody followed by incubation with APC- or APC.Cy7-labelled streptavidin, as described 25 (link). Intracellular staining for Bcl-6, FoxP3 and cytokines was performed using the FoxP3 staining buffer set (eBioscience). Intracellular staining of phospho-S473-AKT (M89-61), pSTAT1 (14/P-STAT1), pSTAT3 (4/P-STAT3) was conducted according to manufacturer’s instructions (BD Bioscience). Cells were acquired on a FACSCantoII using FACSDIva software (BD Biosciences) and analyzed with FlowJo software (Tristar).
Leavenworth J.W., Verbinnen B., Yin J., Huang H, & Cantor H. (2014). A p85α–osteopontin axis couples the ICOS receptor to sustained Bcl-6 expression by follicular helper and regulatory T cells. Nature immunology, 16(1), 96-106.
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2

Multiparameter Flow Cytometry Immunophenotyping

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Mice were sacrificed, then perfused transcardially with Ringer’s solution, and BILs were partially purified as described previously (53 (link)). Cells were incubated with LIVE/DEAD Fixable Dead Cell Stain Kit (Invitrogen, Thermo Fisher Scientific), blocked for Fc receptor binding with 2.4G2, then surface stained with the following Ab: CD45 (30-F11), CD11b (M1/70), CD49d (R1-2), CD62L (Mel14), Ly6G (1A8), Ly6C (HK1.4), CD103 (M290), CD8 (53–6.7), CD4 (GK1.5), PD-1 (29F.1A12), PD-L1 (MIH5), NK1.1 (PK136), MHC-II (I-A/I-E, M5/114.15.2), CD80 (16-10A1), CD86 (GL-1), CD11c (N418), CD3 (145-2C11) and KLRG1 (2F1/KLRG1). For intracellular staining, we used Cytofix/Cytoperm kit (BD Biosciences) and stained for FoxP3 (FJK-16s, BD Biosciences). All Ab were purchased from BioLegend, except CD103 (M290) and CD8 (53-6.7) from BD Biosciences. Stained cells were analyzed on a Gallios flow cytometer (Beckman Coulter) and data analyzed using Kaluza software (Beckman Coulter).
Genoud V., Espinoza F.I., Marinari E., Rochemont V., Dietrich P.Y., McSheehy P., Bachmann F., Lane H.A, & Walker P.R. (2021). Treating ICB-resistant glioma with anti-CD40 and mitotic spindle checkpoint controller BAL101553 (lisavanbulin). JCI Insight, 6(18), e142980.
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3

Multicolor Immunofluorescence Staining Protocol

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Immunofluorescence staining was carried out after acetone fixation, permeabilization, and blocking with Avidin/Protein blocking together with 5% goat serum using rabbit-anti-mouse insulin antibodies (Cell Signaling, 1:100) and donkey-anti-rabbitAlexaFluor647 antibodies (Dianova, 1:400). For CD3 staining, arm.hamster anti-mouse antibodies (BD, 1:50) were used, followed by goat-anti-arm.hamster antibodies conjugated with Dylight488 (Dianova, 1:100). For Tet2 staining, rabbit-anti-Tet2 antibodies (ABclonal, clone A5682, 1:25) was used with biotinylated horse-anti-rabbit antibodies (Vector, 1:100) combined with SADylight 549. For Foxp3 staining, cells were incubated with rat-anti-mouse Foxp3 antibodies (eBioscience, clone FJK-16s, 1:50) and biotinylated goat anti-rabbit (BD), combined with SADylight 549. Nuclei were counterstained with DAPI (Diavona). For Tgfbr1 staining, rat-anti-mouse/human Tgfbr1 antibodies (R&D, clone 141231, 1:25) was used, followed with biotinylated goat-anti-rat antibodies (1:250) combined with SADylight 549 (Dianova, 1:200). Negative control slides were incubated with secondary antibodies. Cells were analyzed by confocal microscopy (Zeiss LSM700).
Scherm M.G., Serr I., Zahm A.M., Schug J., Bellusci S., Manfredini R., Salb V.K., Gerlach K., Weigmann B., Ziegler A.G., Kaestner K.H, & Daniel C. (2019). miRNA142-3p targets Tet2 and impairs Treg differentiation and stability in models of type 1 diabetes. Nature Communications, 10, 5697.
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4

Identifying Regulatory T Cells in Pancreas

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Flow cytometry was performed on a FACSCanto II cytometer (BD Biosciences) using antibodies against Foxp3 (eBioscience, FJK-16s), CD4 (BD Biosciences, RM4-5), and CTLA-4 (eBioscience, UC10-4B9). LIVE/DEAD Fixable Dead Cell Stain Kit (Life Technologies) was used to exclude dead cells. Cells were isolated from the pancreas by digestion with collagenase P. Collected data were analyzed using FlowJo software (Tree Star).
Bengsch F., Knoblock D.M., Liu A., McAllister F, & Beatty G.L. (2017). CTLA-4/CD80 pathway regulates T cell infiltration into pancreatic cancer. Cancer immunology, immunotherapy : CII, 66(12), 1609-1617.
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5

Flow Cytometric Analysis of Th17 and Treg Cells

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For the last four hours of culture, cells were treated with golgi plug (BD), PMA and ionomycin (both from Sigma-Aldrich). Cells were fixed with the FOXP3 fixation buffer (eBioscience) or 4% paraformaldehyde (Sigma-Aldrich), treated with permeabilization buffer (eBioscience) and stained with appropriate antibodies. The antibodies used were: αIL-17a-PE or –APC (eBioscience clone eBio17B7 or BD Biosciences clone N49-653) and αFOXP3-APC or –FITC (eBioscience clone FJK-16s or BD Biosciences clone 259D/C7).
Steinmeyer S., Howsmon D.P., Alaniz R.C., Hahn J, & Jayaraman A. (2017). Empirical Modeling of T cell Activation Predicts Interplay of Host Cytokines and Bacterial Indole. Biotechnology and bioengineering, 114(11), 2660-2667.
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6

Flow Cytometry Analysis of Immune Cells

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Flurochrome-conjugated or biotinylated antibodies against mouse CD4 (RM4-5), CD8 (53-6.7), Foxp3 (FJK-16s), IFN-γ (XMG1.2), IL-4 (BVD6-24G2), NK1.1 (PK136), TCRβ (H57-595), CD45 (30-F11), CD44 (IM7), CD62L (MEL-14), CD11b (M1/70), B220 (RA3-6B2), XCR1 (ZET), NKp46 (29A1.4), CD11c (N418), Ly6c (AL-21), Ly6G (1A8), MHC-II I-A/I-E (M5/114.15.2) were purchased from BD Biosciences, TonBo, eBioscience, Invitrogen and BioLegend. All antibodies were tested with their respective isotype controls. Cell-surface staining was conducted by incubating cells with antibodies for 30 min on ice in the presence of 2.4G2 mAb to block FcγR binding. For Foxp3 staining, a transcription factor-staining kit (Tonbo Biosciences) was used. To assess cytokine production, T cells were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (Sigma), 1 mM ionomycin (Sigma) in the presence of Golgi-Stop (BD Biosciences) for 4 hr at 37°C as previously described35 . T cells were subsequently stained for cell surface markers before intracellular cytokine staining. All data were acquired using an LSRII flow cytometer (Becton Dickinson) and analyzed with FlowJo software (Tree Star, Inc.).
Li S., Liu M., Do M.H., Chou C., Stamatiades E.G., Nixon B.G., Shi W., Zhang X., Li P., Gao S., Capistrano K.J., Xu H., Cheung N.K, & Li M.O. (2020). Cancer Immunotherapy via Targeted TGF-β Signaling Blockade in TH Cells. Nature, 587(7832), 121-125.
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7

Multiparameter Flow Cytometry Analysis

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Cells were stained using antibodies against surface markers CD3 (145-2C11), CD4 (RM4-5) and CD25 (7D4) (all from eBioscience or BD Biosciences), permeabilized, fixed and labeled for intracellular Foxp3 (FJK-16s), IL-4 (11B11), IFNγ (XMG1.2) and IL-17 (eBio17B7). Samples were acquired on LSRII flow cytometer with use of FACSDiva software (BD Biosciences). Data were analyzed using FlowJo software (TreeStar).
de Almeida Nagata D.E., Ting H.A., Cavassani K.A., Schaller M.A., Mukherjee S., Ptaschinski C., Kunkel S.L, & Lukacs N.W. (2015). Epigenetic control of Foxp3 by SMYD3 H3K4 histone methyltransferase controls iTreg development and regulates pathogenic T cell responses during pulmonary viral infection. Mucosal immunology, 8(5), 1131-1143.
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8

Multiparametric Flow Cytometry Immunophenotyping

Cited 1 time
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Fluorescence dye labeled Abs specific for CD4 (L3T4), B220 (RA3-6B2), CD44 (IM7), Fas (15A7), IgM (II/41), T- and B-cell activation antigen (GL7), ICOS (C398.4A), PD-1 (J43), IFNγ (XMG1.2), IL-4 (11B11), IL-17A (eBio17B7), IL-21 (FFA21), Bcl-6 (K112-91) and FoxP3 (FJK-16s) were purchased from BD, eBioscience and Biolegend. Analysis of CXCR5 expression was performed using a biotinylated anti-CXCR5 (2G8, BD) antibody followed by incubation with APC- or APC.Cy7-labelled streptavidin, as described 25 (link). Intracellular staining for Bcl-6, FoxP3 and cytokines was performed using the FoxP3 staining buffer set (eBioscience). Intracellular staining of phospho-S473-AKT (M89-61), pSTAT1 (14/P-STAT1), pSTAT3 (4/P-STAT3) was conducted according to manufacturer’s instructions (BD Bioscience). Cells were acquired on a FACSCantoII using FACSDIva software (BD Biosciences) and analyzed with FlowJo software (Tristar).
Leavenworth J.W., Verbinnen B., Yin J., Huang H, & Cantor H. (2014). A p85α–osteopontin axis couples the ICOS receptor to sustained Bcl-6 expression by follicular helper and regulatory T cells. Nature immunology, 16(1), 96-106.
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9

Comprehensive Immune Cell Profiling

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Single cell suspensions were blocked with Fc-Block (BioXCell) in FACS buffer and surface stained with the following monoclonal antibodies: CD4 (Biolegend, RM4-5), CD8 (eBioscience, 53-6.7), CD19 (6D5, Biolegend), CD25 (BD, PC61), CD44 (eBioscience, IM7), CD62L (MEL-14, eBioscience), CD90.2 (eBioscience, 53–2.1), γδTCR (eBioscience, eBioGL3), Gr-1 (BD, RB6-8C5), F4/80 (eBioscience, BM8). To exclude dead cells, cells were also stained with the fixable viability dye ef780 (eBioscience).
For Treg stainings, cells were fixed and permeabilized according to the PE anti-mouse/rat Foxp3 staining set (eBioscience) and intracellularly stained for Foxp3 (eBioscience, FJK-16s) and CTLA4 (BD, UC10-4F10-11). The cells were acquired with the FACS Canto II (BD Pharmingen) and analyzed with FlowJo Version 8.87. Cells were usually pregated for a lymphocyte gate, singlets and living cells.
Lacher S.M., Bruttger J., Kalt B., Berthelet J., Rajalingam K., Wörtge S, & Waisman A. (2017). HMG-CoA reductase promotes protein prenylation and therefore is indispensible for T-cell survival. Cell Death & Disease, 8(5), e2824-.
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10

Cytokine Production and Apoptosis in CD4+ T Cells

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Where indicated, CD4+ cells were stimulated with PMA (50 ng/mL; Sigma) and Ionomycin (750 ng/mL; EMD Millipore) for 5 h in the presence of GolgiPlug (BD Biosciences). Intracellular staining was performed as previously described(86 (link)). LIVE/DEAD Fixable near-IR Dead Cell Stain (Invitrogen) was used to exclude dead cells. Staining of CD4+ T cells was performed with antibodies to CD4 (RM4–5), IL-6Rα (D7715A7), IL-17A (TC11–18H10), TCR-β (H57–597) and Foxp3 (FJK-16s) from BD Biosciences, IFN-γ (XMG1.2), TCR-β (H57–597), gp130 (KGP130), and Thy1.1 (His51) from eBioscience, and Thy1.1 (OX-7) from Biolegend. For apoptosis analysis, annexin V and propidium iodide staining was performed using an Annexin V Apoptosis Detectin Kit (eBioscience) according to manufacturer’s instructions. Samples were acquired on an LSRII or Attune NxT flow cytometer and data were analyzed with FlowJo software (Tree Star).
Harbour S.N., DiToro D.F., Witte S.J., Zindl C.L., Gao M., Schoeb T.R., Jones G.W., Jones S.A., Hatton R.D, & Weaver C.T. (2020). Th17 Cells Require Ongoing Classic IL-6 Receptor Signaling to Retain Transcriptional and Functional Identity. Science immunology, 5(49), eaaw2262.
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