Fc receptors were blocked by incubation with human gamma globulin (Sigma–Aldrich, St. Louis, MO, USA) and extracellular Ags of interest were stained by incubation with fluorochrome-labeled Abs (Table 2 ) for 30 min on ice. All antibodies were used at an optimized working concentration of 4 μg/ml. PIR-A/B was conjugated to PE/Cy5 using Abcam PE/Cy5 Conjugation Kit according to the manufacturer’s instructions (Abcam, Cambridge, MA, USA). Cells stained with the matched concentrations of relevant isotype Abs were used to determine background binding. Stained cells were acquired on a BD LSR Fortessa and subsequently analyzed using FlowJo V10 (Treestar, Ashland, OR, USA). Due to niche variation in background levels determined by unstained and isotype controls, gates for each Ag analyzed were adjusted on a niche-to-niche basis. This must be taken into consideration when comparing Ag levels on eosinophils recovered from different niches. Geometric mean fluorescence intensity was used to compare expression of Ags in different groups within the same niche, with isotype control fluorescence subtracted mathematically. Samples where negative values resulted from subtracting the isotype control fluorescence were designated a value of 0. Up to 1 outlier per group were eliminated using Grubb’s test if deemed an outlier.
Human gamma globulin
Manufactured by Merck Group
Sourced in United States
Human gamma globulin is a blood plasma fraction that contains immunoglobulins, primarily immunoglobulin G (IgG). It is used as a laboratory reagent for various immunological and diagnostic assays.
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Lab products found in correlation
Lsrfortessa, by BD (2 mentions)
Fluoromount g, by Thermo Fisher Scientific (1 mentions)
Bz 9000, by Keyence (1 mentions)
Facsdiva software, by BD (1 mentions)
Facsaria 3 flow cytometer, by BD (1 mentions)
Anti cd271 antibody, by Lab Vision (1 mentions)
Collagenase 4, by Merck Group (1 mentions)
Phalloidin alexafluor647, by Thermo Fisher Scientific (1 mentions)
Jasplakinolide, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 antibody, by Thermo Fisher Scientific (1 mentions)
Golgiplug, by BD (1 mentions)
Gentlemacs, by Miltenyi Biotec (1 mentions)
Donkey serum, by Merck Group (1 mentions)
7 protocols using human gamma globulin
1
Surface Protein Expression Profiling
2
Immunofluorescence Analysis of PEDF-R and Laminin Receptor
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Cells plated on coverslips were fixed with 1% paraformaldehyde for 30 min on ice. After washing with TBS, cells were blocked with TBS/1% dimethyl sulfoxide/0.3% Triton X-100/3% BSA, 2 mg/mL human gamma globulin (Sigma-Aldrich) for 1 h, and incubated with antibodies directed to PEDF-R (mouse anti-ATGL, Cat# sc-365278; Santa Cruz) or LR (rabbit anti-laminin receptor, Cat# ABC934; Merck Millipore, Darmstadt, Germany) at 4 °C overnight. After washing in TBS, coverslips were incubated at room temperature with Cy3-labeled goat anti-mouse IgG or anti-rabbit IgG for 2 h. Cells were washed, counterstained with DAPI and mounted with Fluoromount-G (Thermo Fisher Scientific). Negative control stains were included using mouse IgG1 and normal rabbit IgG, respectively, at concentration similar to that of PEDF-R and LR antibodies. Images were taken using a fluorescence microscope (BZ-9000, Keyence, Osaka, Japan).
3
Isolation and Characterization of CD271+ Cells
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To obtain CD271-positive cells, normal human keratinocytes were incubated in blocking buffer containing DMEM, 10% FBS, 0.1 M sodium azide, and 4% human gamma globulin (Sigma, St. Louis, MO) for 20 min on ice. After staining cells with 0.5% bovine serum albumin (BSA) in PBS with anti-CD271 antibody (1:100 in PBS, Lab Vision Corporation) at room temperature (RT) for 15 min, cells were resuspended with Alexa Fluor anti-mouse antibody (Invitrogen, Paisley, UK) in 0.5% BSA in PBS for 15 min at room temperature. Following this, cell viability solution (7-AAD solution, BD Via-Probe™, Biosciences Pharmingen, San Diego, CA, USA) was added to the pellet of cells for 15 min at room temperature. Finally, culture medium was added to cells. Negative control was obtained by omitting the primary antibody. Data were collected using a FACS Aria III flow cytometer (BD Biosciences) and analyzed on FACS Diva software (BD Biosciences).
4
Multicolor Flow Cytometry Analysis of Lymphocytes
5
Integrin Expression Analysis by Flow Cytometry
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Integrin expression was monitored by flow cytometry and Geometric Mean Fluorescence Intensity (GMFI) was determined and normalized against the GMFI obtained for isotype controls. Staining was performed in a p96 U-bottom plate (Thermo Scientific) Cells were fixed with 2% formaldehyde for 10 minutes and blocked for 30 minutes with a PBS buffer containing 1% BSA and 100 μg/ml human gamma globulin (Sigma). When required, the cells were permeabilized with 0.1% Triton X-100 in PBS buffer for 10 minutes before blocking. The following hybridoma-derived monoclonal mouse antibodies were used to detect protein expression: BEAR-1 (integrin αM) (24 (link)), Ts1/11 (integrin αL) (25 (link)), HC1/1 (integrin αX) (26 (link)), BU15 mAb anti-CD11c (Immunotools), Ts2/16 (integrin β1) (27 (link)), Lia2/3 (integrin β2) (28 (link)), PAINS-10 (tetraspanin CD9) (29 (link)), Vj1/12 (CD59) (30 (link)) and 5A6 (tetraspanin CD81) (31 (link)). Cells were washed and stained with a donkey anti-mouse Alexa Fluor® 488 antibody (Life Technologies), as per the manufacturer’s instructions. To determine total cellular F-actin, Phalloidin-Alexa Fluor® 647 (Life Technologies) was used. When indicated, cells were treated with 1 µM jasplakinolide (Santa Cruz) 24 hours previous to completing the retinoic acid-induced differentiation.
6
Multiparametric Flow Cytometry of Murine Lungs
7
Quantifying CD47 Expression on CD45+ Cells
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Skin OCT sections of 5 μm were fixed (formaldehyde 4%), permeabilized (Triton X-100 0,2%) and blocked with 100 μg/ml human gammaglobulin (Sigma-Aldrich, St. Louis MO, USA) and a 1:100 dilution of donkey serum (Sigma-Aldrich) in phosphate buffer solution (PBS). Skin sections were then incubated over-night with 5 μg/ml sheep anti-human CD47 (R&D systems, Cat. AF4670) and mouse anti-human CD45, followed by donkey anti-sheep (DAS) Alexa Fluor 488 and donkey anti-mouse (DAM) Alexa Fluor 555. Finally, cell nuclei were counterstained with DAPI. Negative controls were performed with omission of the primary antibody. Sections were examined with a Leica DMR immunofluorescence microscopy under the same acquisition conditions. Images were analyzed using the ImageJ sowftware (http://imagej.softonic.com ). For the analysis of CD47 expression, fluorescence intensity was determined in regions of interest (ROIs) drawn on CD45+ cells.
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