Monocyte-derived macrophages were infected with the R5-tropic HIV and the infection was left to progress. At day 11, infection percentage was evaluated by p24 intracellular staining as described in the following paragraph (Figure S1 in Supplementary Material). After that, MDMs were washed twice with PBS 1× (Sigma) and rhMIF was added to a final concentration of 1, 10, or 25 ng/ml. Cells were incubated at 37°C for 8 h until the supernatant was collected. When denoted, pretreatment with the αCD74 blocking antibody (or the appropriate isotype control) was performed at 5 ng/ml for 30 min. In some experiments (TLR4 expression), Fc receptors were blocked for 10 min before the addition of the αCD74 blocking antibody (or its isotype-matched control) with an Fc blocking reagent from BD Biosciences.
Fc blocking reagent
Manufactured by BD
Sourced in United States, Germany
The Fc blocking reagent is a laboratory product designed to prevent non-specific binding of antibodies to Fc receptors. It is a solution that can be added to samples or assays to block Fc-mediated interactions, ensuring accurate and reliable results in immunoassays and other applications.
Automatically generated - may contain errors
Lab products found in correlation
Facsaria cell sorter, by BD (3 mentions)
Zombie red fixable, by BioLegend (2 mentions)
Celltrace cfse cell proliferation kit, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Rbc lysis buffer, by Thermo Fisher Scientific (2 mentions)
Pbs 1, by Merck Group (1 mentions)
Fortessa flow cytometer, by BD (1 mentions)
Fluorescein isothiocyanate (fitc), by BD (1 mentions)
Gallios cytometer, by Beckman Coulter (1 mentions)
Facscan instrument, by BD (1 mentions)
Cd11b percp cy5, by BioLegend (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Kaluza software, by Beckman Coulter (1 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Facsdiva software v6, by BD (1 mentions)
Cd11b pe cy5, by BD (1 mentions)
10 protocols using fc blocking reagent
1
Investigating HIV Infection in Macrophages
2
Isolation and Flow Cytometric Analysis of Brain Microglia
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Mononuclear cells were isolated via a density centrifugation technique at the interface of a 30/70% Percoll gradient (Fisher Scientific #17-5445-01), as previously described [28 (link)]. Cells were blocked using Fc blocking reagent (BD Biosciences #553141) for 10 min before a 30-min incubation with fluorophore-conjugated flow cytometry antibodies against CD11b (FITC; BD Biosciences #553310), CD45 (APC; BD Biosciences; #559864), and CX3CR1 (PE; BD Biosciences #565798). Data were acquired using BD Biosciences Fortessa Flow Cytometer and analyzed using FlowJo single cell analysis software. Fifty thousand events were minimally collected before data processing. Mean fluorescent intensity was utilized in conjunction with total event counts in order to quantify the number of brain-resident microglia (CD11b+/CD45low cells) and relative expression of CX3CR1.
3
Flow Cytometry Analysis of Cell Proliferation and Apoptosis
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For flow cytometeric analysis, cells were washed with FACS buffer (1 × PBS, 2.5% FBS), resuspended in 100 μl FACS buffer, incubated with an Fc blocking reagent (BD PharMingen) and stained at 4∘C with antibodies to various cell surface antigens. Viability was assessed with DAPI (Invitrogen) or Zombie red fixable (BioLegend). Cells were washed 1× with FACS buffer, and resuspended in 200 μl FACS buffer. Flow cytometric analysis of CFSE or Annexin V stainings was performed according to manufacturer’s instructions (CellTrace CFSE Cell Proliferation Kit, Invitrogen and FITC Annexin V Apoptosis Detection Kit, BD Biosciences. Briefly, purified naive B cells were stained with 5 μM CFSE and cultured for 96 h in LPS plus IL-4 and CFSE dilution was monitored by flow cytometer. GFP+ cells or bone marrow B cell populations were sorted in a FACSAria cell sorter (BD Biosciences). Flow cytometry was performed using an LSR-II flow cytometer (BD Biosciences), and data analyzed using FlowJo software (version 9.9).
4
Flow Cytometry Analysis of Myeloid Cells
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Single-cell suspension obtained from spleen, BM and PB isolated from NB-bearing and healthy animals were washed with PBS (Sigma), incubated with Fc blocking reagent (BD Biosciences, San Diego, CA, USA) and then stained for 20 min (min) at 4 °C with the relevant Ab. The Abs used were: CD11b PerCP/Cy5.5 (BioLegend, London, UK), Ly-6G/Ly-6C (Gr-1) APC (BioLegend) and isotype-matched control monoclonal antibodies (Biolegend). After staining, samples were acquired with a Gallios cytometer (Beckman Coulter, Milan, Italy) and data were analyzed using Kaluza software (Beckman Coulter).
In some experiments, MSC-1 and MSC-2 cell lines were pretreated or not with LPS (Sigma) (1 μg/ml) for 4 h and then treated with BzATP (100 and 300 μM) for 30 min. The cells were fixed with 2% paraformaldehyde at RT for 20 min and permeabilized with permeabilization buffer (PBS, 1% FBS, 0.1% saponin, Sigma). Then, cells (5 × 105/tube) were incubated with anti-ARG-1 polyclonal Ab from Sigma and Santa Cruz Biotechnology (Heidelbergh, Germany) for 30 min at RT, washed twice with permeabilization buffer and incubated with FITC-conjugated F(ab')2 fragments of goat anti-rabbit IgG antibodies (Abcam, Cambridge, UK). After resuspension in staining buffer, cells were then analyzed by flow cytometry using a FACScan instrument (BD Biosciences, San Josè, CA, USA).
In some experiments, MSC-1 and MSC-2 cell lines were pretreated or not with LPS (Sigma) (1 μg/ml) for 4 h and then treated with BzATP (100 and 300 μM) for 30 min. The cells were fixed with 2% paraformaldehyde at RT for 20 min and permeabilized with permeabilization buffer (PBS, 1% FBS, 0.1% saponin, Sigma). Then, cells (5 × 105/tube) were incubated with anti-ARG-1 polyclonal Ab from Sigma and Santa Cruz Biotechnology (Heidelbergh, Germany) for 30 min at RT, washed twice with permeabilization buffer and incubated with FITC-conjugated F(ab')2 fragments of goat anti-rabbit IgG antibodies (Abcam, Cambridge, UK). After resuspension in staining buffer, cells were then analyzed by flow cytometry using a FACScan instrument (BD Biosciences, San Josè, CA, USA).
5
Flow Cytometry Analysis of Cell Proliferation and Apoptosis
6
Murine Immune Cell Isolation and Analysis
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Blood were collected and mixed with RBC lysis buffer (eBioscience, Carlsbad, CA, USA) to lyse red blood cells for 5 min. The same volume of PBS was applied to stop the RBC lysis reaction, and the cell pellet was resuspended in PBS. Cell suspensions were blocked with 1% goat serum (Gibco) and 0.2% Fc blocking reagent (BD Pharmingen) for 30 min. After blocking, cells were stained with fluorescence conjugated antibodies against CD11b, Ly6C, Ly6G, or CD45 (BD Phamingen) for 30 minutes on ice. The cell suspensions were washed twice by PBS before analysis on a BD FACSCantoTM flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and data were analyzed by FACSDiva software v6.1.3 (Becton Dickinson).
7
Isolation and Identification of Myeloid-Derived Suppressor Cells from Infected Murine Cardiac Tissue
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Cell suspensions were obtained from digested cardiac tissue samples of infected mice treated with MSCs, MSC_G-CSF, or saline, as previously described (16 (link)). The cell suspensions were allowed to pass through a 70 µm cell strainer (BD Biosciences, Franklin Lakes, NJ, USA) and counted with a hematocytometer. Aliquots of 106 cells were used for each test tube and 1 µL of Fc blocking reagent (BD Biosciences) was added. Fluorochrome-conjugated antibodies used were: CD11b-PE-Cy5, CD45-APC and GR-1-FITC, or Ly6C-FITC and Ly6C-PE (BD Biosciences). Samples were incubated with the antibodies for 20 min at RT. Sample acquisition was performed using a BD LSRFortessa SORP cytometer using BD FacsDiva v.6.2. Acquired data were analyzed by FlowJo v7.5 (FlowJo Enterprise, Ashland, OR, USA). CD11b+GR-1+ MDSCs were sorted from digested hearts of MSC_G-CSF-treated mice or from the bone marrow, as indicated in Section “Results .” The cells were stained with GR-1-PE (BD Biosciences), CD11b-APC (ThermoFisher Scientific), and CD45-APC-Cy7 (BD Biosciences), using a FACS Aria cell sorter (BD Biosciences), achieving a purity of approximately 98%.
8
Single-Cell Sorting of Primary Pancreatic Tumors
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For fresh primary pancreatic tumor tissue, single cell suspension was made by mechanical and enzymatic dissociation. Cells were blocked with Fc blocking reagent (BD Bioscience). After labeling of Live/Dead dye, cells were stained with EpCAM-PE (Biolegend) and FAP-APC (R&D) on ice for 30 min for sorting (BD Aria II). The gating was based on the isotype staining.
9
Quantifying T2R10 Expression by Flow Cytometry
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For flow cytometry analyses ~1x106 cells were harvested with Accutase (Life technologies, Darmstadt, Germany) and resuspended in 100 µl flow cytometry buffer (FACS buffer: PBS; 0.5% FCS) followed by fixation in ice-cold Methanol (80%) for 20 minutes at 4°C. Then, cells were washed with flow cytometry buffer and permeabilized with PBS plus 1% Tween 20 (Gerbu Biotechnik, Heidelberg, Germany) for 20 minutes at room temperature. After washing with FACS-buffer containing 0.1% Tween 20, cells were pre-incubated with 10 µl Fc Blocking Reagent (Becton and Dickinson, Heidelberg, Germany) for 15 minutes. Then the primary antibody against T2R10 (ab138285, abcam, Cambridge, UK) (20 ng/ml) and IgG isotype control (20 ng/ml) were incubated for 35 minutes, respectively. Cells were washed and incubated with a PE-labeled secondary antibody (5 ng/ml) for 20 minutes protected from light. Finally, cells were washed and resuspended in 300 µl buffer. All incubation steps were performed at room temperature. BD FACS Canto 2 was used for detection. Results were analyzed with FlowJo 10.0.8 software (Tree Star, Ashland, OR, USA). The mean fluorescence intensity (MFI) was used to show expression. In the knock down experiments, the MFI of cells treated with siRNA negative control was defined as 100%. Knockdown efficacy was expressed as percentage decrease of MFI.
10
Myeloid Cell Trafficking to Tumors
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5 × 106 CFDA-labelled CD11b+ myeloid cells purified by gradient centrifugation from male and female 6–8-week-old C57BL/6 WT and Mlck210−/− mice in the C57BL/6 background or 5 × 106 CFDA-labelled Mlck and non-silencing siRNA-transfected CD11b+ cells were injected intravenously into WT mice bearing subcutaneous d14 LLC tumors. Animals were euthanized 2 or 24 h after myeloid cell injection. Fluorescent cells accumulating in tumors and spleens were quantified at 2 h and 24 h after inoculation by flow cytometry of single-cell preparations of tumors. To quantify inoculated CDFA+ myeloid cells in tissues, tumors were excised, minced, and digested to single-cell suspensions for 1 h at 37 °C in 5 ml of Hanks Balanced Salt Solution (HBSS, GIBCO) containing 1 mg/ml Collagenase type IV, 10 µg/ml Hyaluronidase type V and 20 units/ml DNase type IV (Sigma). Red blood cells were solubilized with RBC Lysis Buffer (ThermoFisher). Single-cell suspensions (106 cells in 100 µL total volume) were incubated with 0.5 µg/ml propidium iodide and then with Fc-blocking reagent (Becton Dickinson) or serum. Cells were gated on SSC-A vs FSC-A to identify cells, then gated on PI negative populations to identify live cells and then on FITC vs SSC-A to identify and quantify green-fluorescent myeloid cells. Facs analysis was performed using FloJo software (Treestar, Inc).
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