PBMCs were stained with FITC-labeled anti-CD19 antibody (or isotype IgG) (BD Bioscience) for 30 min at 4°C, washed with phosphate-buffered saline (PBS), fixed with 1% paraformaldehyde for 1 h, incubated with 0.5% saponin (Sigma Aldrich), stained with APC-labeled anti-IL-10 antibody (or isotype IgG) for 30 min at 4°C, washed with PBS, and analyzed with a flow cytometer (FACSCanto II; BD Bioscience). The data were analyzed with Flowjo (TreeStar, Ashland OR). Data from isotype IgG-stained cells were used as a gating reference.
Isotype igg
Manufactured by BD
Isotype IgG is a type of antibody used in various laboratory techniques. It serves as a control to establish baseline signals and normalize experimental data.
Automatically generated - may contain errors
Lab products found in correlation
Saponin, by Merck Group (1 mentions)
Facscanto 2, by BD (1 mentions)
Cd45 pe cy7, by BD (1 mentions)
Cell lab quanta sc, by Beckman Coulter (1 mentions)
Cd34 pe cy5, by BD (1 mentions)
Cd73 pe, by BD (1 mentions)
Pe conjugated mouse anti human cd73, by BD (1 mentions)
Ab19857, by Abcam (1 mentions)
Fitc conjugated goat anti rabbit ig, by Merck Group (1 mentions)
Isotype igg, by BioLegend (1 mentions)
H2 kb, by BD (1 mentions)
Accuri c6, by BD (1 mentions)
5 protocols using isotype igg
1
Flow Cytometric Analysis of IL-10 in PBMCs
2
Characterizing NPC Phenotype by Flow Cytometry
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The phenotype of NPCs was determined by cytofluorimetric analysis with fluorochrome‐conjugated antibodies against human CD90‐FITC (#555595) at 1:100, CD73‐PE (#550257) at 1:100, CD105‐Alexa488 (#MHCD10520) at 1:100, CD34‐PECy5 (#555823) at 1:100, CD45‐PECy7 (#557748) at 1:100, CD146‐BV605 (#7433019) at 1:100, and Tie2‐PE (#FAB3131A) at 1:50 (all from BD Bioscience, Franklin Lakes, NJ). Isotype IgG was used as a control (all from BD Biosciences). Cells in suspension were incubated for 40 minutes with each of these antibodies at 4°C in FACS buffer (PBS, 0.5% human serum albumin, 0.5 mm EDTA), and analyzed with a Cell Lab Quanta SC flow cytometer (Beckman Coulter Inc.).
3
Mesenchymal and Hematopoietic Cell Markers Analysis
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Evaluation of the expression of mesenchymal stem cell surface markers (CD73 and CD44), embryonic stem cell surface marker (OCT-4), and hematopoietic cell marker (CD45) was done by flow cytometric analysis as described previously [28 (link)]. Cells (105 cells/100 μl) were gently washed in PBS containing 2% FBS and incubated separately with PE-conjugated mouse anti-human CD73 (561014; BD Pharmingen), CD44 (550989; BD Pharmingen), and CD45 (560975; BD Pharmingen) in darkness for at least 40 min at 4 °C. Evaluation of OCT-4 expression was analysed using indirect intracellular flow cytometry. The cells were permeabilized with 0.1% saponin. Then primary rabbit antihuman OCT-4 antibody (ab19857, Abcam) was added for 40 min and then incubated with FITC-conjugated goat anti-rabbit Ig (Sigma-Aldrich) for 30 min. As negative controls, isotype IgG (555748; BD Pharmingen) was used. Afterward, cells were washed twice with PBS-FBS, fixed in 1% formaldehyde solution, and analyzed by a flow cytometer (Partec GmbH).
4
NK Cell Cytotoxicity Assay with Red-A
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A549, H1299 and NK cells were seeded into six-well plates in the presence or absence of 10–1000 nM of Red-A for 24 h. The pre-treated A549 or H1299 cells were cultured with NK cells at 1:1 ratio while the pre-treated NK cells were incubated in the presence or absence of A549 or H1299 cells at 1:1 ratio. Next, cells were stained with PE/Cy5-conjugated CD107α antibody (Biolegend, San Diego, CA, 555802) or isotype IgG (Biolegend, San Diego, CA, 409304) and incubated at 37 °C for 4 h, following by FITC-conjugated anti-human CD56 antibody (BD Biosciences, Franklin Lakes, NJ, 308304) or isotype IgG (BD Biosciences, Franklin Lakes, NJ, 551497) at 4 °C for 30 min, then the percentage of CD56+/CD107α+ NK cells was assessed by flow cytometry (BD Biosciences, Franklin Lakes, NJ, Accuri C6).
5
Multiparametric Flow Cytometry Assay
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Fluorescence‐conjugated antibodies against mouse CD45, CD8a, CD314, NK1.1, H‐2Kb molecules and isotype IgG (BD Biosciences), the antibodies against mouse Rae1, H60, MULT1, isotype IgG (R&D Systems) and Alexa Fluor 647 goat anti‐rat (Bioss) secondary antibodies were used. All stained cells were analyzed using Accuri C6 (BD Biosciences).
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