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Clone 93

Manufactured by Thermo Fisher Scientific
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The Clone 93 is a laboratory instrument designed for cell culture applications. It provides an efficient and controlled environment for the growth and maintenance of cell lines. The core function of the Clone 93 is to facilitate the cultivation of cells in a temperature and CO₂-regulated setting, ensuring optimal conditions for their proliferation and differentiation.

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Lab products found in correlation

Fvd efluor 506, by Thermo Fisher Scientific (3 mentions) Ack lysing buffer, by Thermo Fisher Scientific (2 mentions) X20 cytometer, by BD (2 mentions) Trucount beads, by BD (2 mentions) Imiquimod, by InvivoGen (1 mentions) Clone m1 70, by Thermo Fisher Scientific (1 mentions) Anti mouse cd16 cd32, by Thermo Fisher Scientific (1 mentions) Clone rb6 8c5, by Thermo Fisher Scientific (1 mentions) Clone bm8, by Thermo Fisher Scientific (1 mentions) Facscalibur, by BD (1 mentions) Clone 30 f11, by Thermo Fisher Scientific (1 mentions) Facsaria 3 4l, by BD (1 mentions) Ter119 pe, by Thermo Fisher Scientific (1 mentions) Cd45 pe, by Thermo Fisher Scientific (1 mentions) Cd44 pe cy7, by Thermo Fisher Scientific (1 mentions) Sca 1 percp cy5, by Thermo Fisher Scientific (1 mentions) Cd90.2 efluor 450, by Thermo Fisher Scientific (1 mentions) Gp38 apc, by Thermo Fisher Scientific (1 mentions) Clone 8, by Thermo Fisher Scientific (1 mentions) Cd31 pe, by Thermo Fisher Scientific (1 mentions)

6 protocols using clone 93

1

Isolation and Stimulation of Plasmacytoid Dendritic Cells

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Splenic and bone-marrow-derived pDCs were isolated and after blockade of Fcγ receptors with anti-CD16/32 (eBioscience; clone 93), cells were stained for viability (eBioscience; FVD eFluor 506) and with B220 BV711, PDCA-1 FITC, Siglec H APC, and LyC6 Pacific. Primary cells were cultured in complete RPMI 1640 with 10% fetal bovine serum (FBS), 1% glutamax, 1% nonessential amino acids, 1% sodium pyruvate, 1% penicillin-streptomycin, and 1 uM β-mercaptoethanol (Gibco-Invitrogen). Sorted pDCs were stimulated overnight with CpGA 2216 (6 μg/ml, Operon); CpGB 1826 (6 μg/ml, Operon) or Imiquimod (6 μM, InvivoGen). IL-6 and IFN-α levels were measured by ELISA in the cell supernatant at 18 h post-stimulation.
Winkler E.S., Shrihari S., Hykes BL J.r., Handley S.A., Andhey P.S., Huang Y.J., Swain A., Droit L., Chebrolu K.K., Mack M., Vanlandingham D.L., Thackray L.B., Cella M., Colonna M., Artyomov M.N., Stappenbeck T.S, & Diamond M.S. (2020). The intestinal microbiome restricts alphavirus infection and dissemination through a bile acid-type I IFN signaling axis. Cell, 182(4), 901-918.e18.
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2

Multicolor Flow Cytometry Analysis of Pancreatic Immune Cells

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Pancreatic cells isolated from each group were washed by a fresh FACS buffer for three times, then cell suspensions were treated with the anti-mouse CD16/CD32 (1:100 ratio, clone 93, eBioscience, Santa Clara, CA, USA) for 15 min to block the Fc receptor. Cell suspensions were subsequently labeled with anti-mouse PE-Cyanine 5 conjugated CD11b (1:500 ratio, clone M1/70, eBioscience, Santa Clara, CA, USA) and anti-mouse FITC conjugated F4/80 (1:500 ratio, clone BM8, eBioscience, Santa Clara, CA, USA), and/or anti-mouse PE conjugated Ly6G (1:500 ratio, clone RB6-8C5, eBioscience, Santa Clara, CA, USA) for 30 min on ice in the dark to detect macrophages or neutrophils. Stained cells were measured with the FACS Calibur (Becton Dickinson, San Jose, CA, USA) and flow cytometric data were analyzed using CellQuestPro software.
Zhou Z., Choi J.W., Shin J.Y., Kim D.U., Kweon B., Oh H., Kim Y.C., Song H.J., Bae G.S, & Park S.J. (2021). Betulinic Acid Ameliorates the Severity of Acute Pancreatitis via Inhibition of the NF-κB Signaling Pathway in Mice. International Journal of Molecular Sciences, 22(13), 6871.
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3

Comprehensive Immune Cell Profiling

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Peripheral blood was obtained from the submandibular vein. Erythrocytes were lysed twice with ammonium chloride-potassium (ACK) lysing buffer (Gibco), and remaining cells were resuspended in PBS supplemented with 2% FBS and 1 mM EDTA and maintained on ice. After blockade of Fcγ receptors with anti-CD16/32 (eBioscience; clone 93) and confirmation of viability (eBioscience; FVD eFluor 506), staining for cell surface antigens CD45 BUV395, CD3 APC, CD19 PE, Ly6G BV650, Ly6C Pacific Blue, CD11b PE/Dazzle 594, NK1.1 FITC, CD4 BV605, and CD8 PerCPCy5.5 was performed at dilutions of 1:200. Cells were incubated for 20 min at 4°C, fixed with 4% PFA for 20 min, and washed prior to resuspension in PBS supplemented with 2% FBS and 1 mM EDTA. Absolute cell counts were determined using TruCount beads (BD Biosciences). Flow cytometry data were acquired on a BD-X20 cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).
Winkler E.S., Bailey A.L., Kafai N.M., Nair S., McCune B.T., Yu J., Fox J.M., Chen R.E., Earnest J.T., Keeler S.P., Ritter J.H., Kang L.I., Dort S., Robichaud A., Head R., Holtzman M.J, & Diamond M.S. (2020). SARS-CoV-2 infection of hACE2 transgenic mice causes severe lung inflammation and impaired function. Nature immunology, 21(11), 1327-1335.
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4

Comprehensive Immune Cell Profiling

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Peripheral blood was obtained from the submandibular vein. Erythrocytes were lysed twice with ammonium chloride-potassium (ACK) lysing buffer (Gibco), and remaining cells were resuspended in PBS supplemented with 2% FBS and 1 mM EDTA and maintained on ice. After blockade of Fcγ receptors with anti-CD16/32 (eBioscience; clone 93) and confirmation of viability (eBioscience; FVD eFluor 506), staining for cell surface antigens CD45 BUV395, CD3 APC, CD19 PE, Ly6G BV650, Ly6C Pacific Blue, CD11b PE/Dazzle 594, NK1.1 FITC, CD4 BV605, and CD8 PerCPCy5.5 was performed at dilutions of 1:200. Cells were incubated for 20 min at 4°C, fixed with 4% PFA for 20 min, and washed prior to resuspension in PBS supplemented with 2% FBS and 1 mM EDTA. Absolute cell counts were determined using TruCount beads (BD Biosciences). Flow cytometry data were acquired on a BD-X20 cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).
Winkler E.S., Bailey A.L., Kafai N.M., Nair S., McCune B.T., Yu J., Fox J.M., Chen R.E., Earnest J.T., Keeler S.P., Ritter J.H., Kang L.I., Dort S., Robichaud A., Head R., Holtzman M.J, & Diamond M.S. (2020). SARS-CoV-2 infection of hACE2 transgenic mice causes severe lung inflammation and impaired function. Nature immunology, 21(11), 1327-1335.
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5

Comprehensive Immune Cell Profiling

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Cells in single cell suspensions were blocked with anti-mouse CD16/CD32 (1:200; Clone 93, Thermo Fisher Scientific) for 10 min at 4°C, followed by incubation on ice for 30 min with the appropriate combination of fluorochrome-conjugated antibodies diluted in FACS buffer: TER119-PE, (1:600, clone TER-11, eBioscience), CD45-PE (1:300, clone 30-F11, eBioscience), CD31-PE (1:300, clone 390, Thermo Fisher Scientific), gp38-APC (1:100, clone 8.1.1, eBioscience), CD90.2-eFluor 450 (1:300, clone 53-2.1, eBioscience), Sca-1- PerCP-Cy5.5 (1:300, clone D7, eBioscience), CD44-PE-Cy7 (1:300, clone IM7, eBioscience), CD140a-PE-Cy7 (1:300, clone APA5, eBioscience). 4′,6-diamidino-2-phenylindole (DAPI 1 μg/mL, Roche) was used to distinguish live/dead cells. Cells were analyzed with the BD LSR Fortessa flow cytometer (BD Biosciences) and sorted with the FACSAria III 4L (BD Biosciences). FlowJo (version 10.08) was used for data analyses.
Stellato M., Czepiel M., Distler O., Błyszczuk P, & Kania G. (2019). Identification and Isolation of Cardiac Fibroblasts From the Adult Mouse Heart Using Two-Color Flow Cytometry. Frontiers in Cardiovascular Medicine, 6, 105.
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6

Multicolor Flow Cytometry of Colon Immune Cells

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All antibodies used for flow cytometry were purchased from either ThermoFisher, BD Biosciences, or BioLegend. The antibodies we used for flow cytometry are: CD45.2 (Invitrogen, clone 104, ref 47-0454-82), EPCAM (BD Biosciences, clone G8.8, catalog # 563478), activated-Caspase-3 (BD Biosciences, clone C92-605, catalog # 560901), and Ki67 (BioLegend, clone 16A8, catalog # 652403). Dead cells were discriminated in all experiments using LIVE/DEAD (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted August 19, 2021. ; https://doi.org/10.1101/2021.08.18.456840 doi: bioRxiv preprint 20 fixable dead stain (ThermoFisher, catalog # 501121526). All stains were carried out in media containing anti-CD16/32 blocking antibody (ThermoFisher, clone 93, catalog # 14-0161-86). All flow cytometry was acquired on an LSRFortessa FACS analyzer. Cells were isolated from the colon for flow cytometry using EDTA and DTT dissociation and shaking to release the epithelium from the lamina propria (Hall et al., 2011) (link). To separate intraepithelial cells, the cell suspension was spun down in a 30% percoll gradient. Analysis of flow cytometry was carried out on FlowJo software (TreeStar).
Burr A.H., Ji J., Ozler K., Eskiocak O., Yueh B., Menk A.V., Costa A.S., Rittenhouse N., Marshall C.W., Chiaranunt P., Mullinax L., Overacre-Delgoffe A., Cooper V.S., Poholek A.C., Delgoffe G.M., Beyaz S., & Hand T.W. (2021). Excess dietary sugar impairs colonic epithelial regeneration in response to damage.
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