Pancreatic cancer cell lines BxPC-3, MIAPaCa-2, and PaCaDD135 were analyzed for the expression of TLR2, -4, and -9 using a flow cytometer (Beckman Coulter, Krefeld, Germany) with a software package Coulter Epics XL-MCL, System II (Beckman Coulter). Phycoerythrin (PE)-conjugated TLR2 and TLR4 antibodies as well as isotype control antibodies were purchased from Miltenyi (Bergisch Gladbach, Germany), PE-conjugated antibody against TLR9 was provided by eBioscience (San Diego, CA, USA). For intracellular staining we used Intraprep-Kit (Beckman Coulter). Staining was performed according to the manufacturers’ instructions.
Isotype control antibody
Manufactured by Miltenyi Biotec
Sourced in Germany
Isotype control antibodies are laboratory reagents used in flow cytometry and other immunoassays to establish the specificity of target-specific antibodies. They are immunoglobulin molecules of the same isotype as the target-specific antibody, but with irrelevant antigen specificity.
Automatically generated - may contain errors
Lab products found in correlation
Intraprep kit, by Beckman Coulter (1 mentions)
Coulter epics xl mcl system 2, by Beckman Coulter (1 mentions)
Anti cd73 pe, by Miltenyi Biotec (1 mentions)
Anti cd44 apc, by Miltenyi Biotec (1 mentions)
Anti cd105 apc, by Miltenyi Biotec (1 mentions)
Accutase, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Facscalibur, by BD (1 mentions)
Facsaria 3 cell sorter, by BD (1 mentions)
Facscanto 2 system, by BD (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Pd l1, by BioLegend (1 mentions)
Cd163, by Santa Cruz Biotechnology (1 mentions)
Cellquest software, by BD (1 mentions)
4 protocols using isotype control antibody
1
Pancreatic Cancer Cell TLR Expression
2
Flow Cytometry-based Sorting of p75NTR+ Cells
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Cells were dissociated by treatment with Accutase (Invitrogen), resuspended in 1% bovine serum albumin (BSA) (Sigma‐Aldrich) in phosphate‐buffered saline (PBS) and incubated for 15 minutes at 4°C with anti‐CD271 (p75NTR)‐PE, anti‐SOX1‐PE, anti‐CD44‐APC, anti‐CD73‐PE or anti‐CD105‐APC (all from Miltenyi Biotec, Bergisch Gladbach, Germany). The cells were washed once in 1% BSA in PBS and analysed using a BD FACSCalibur (BD Biosciences). Isotype control antibodies (Miltenyi Biotec) were used as negative controls. p75high and p75low cells were separately sorted using BD FACSAria™ III Cell Sorter (BD Biosciences).
3
Identifying Apoptosis in Pancreatic Cancer
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After specific treatment, pancreatic cancer cells were stained with fluorochrome-conjugated monoclonal antibodies for CD133 (Miltenyi Biotec, Teterow, Germany) or an isotype control antibody (Miltenyi Biotec). Apoptosis assay was performed using the Annexin V FITC Apoptosis Detection KIT (BD Biosciences). Flow cytometric analysis was performed on a BD FACSCanto II system (BD Biosciences).
4
Flow cytometric analysis of macrophages
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After 24 h, uninfected, KSHV-infected or UV-KSHV treated macrophages were stained with antibodies against CD86 (Miltenyi Biotec, 130-094-877), CD163 (Santa Cruz Biotechnology, sc20066), PD-L1 (Biolegend, 329706) and isotype control antibody (Miltenyi Biotec, 130-095-897) and analysed by FACS Calibur flow cytometer (BD Transduction Laboratories), using CELLQuest software (BD Biosciences).28 (link) Debris and dead cells were excluded from the analysis, gating live cells in a forward versus side scatter (FSC vs SSC) density plot. For each analysis 10.000 events were recorded.
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