The isolation of lymphocytes from spleen, peripheral/mesenteric lymph nodes, small and large intestinal lamina propria, and small intestine intraepithelial lymphocytes were performed as previously described.71 (link) After digestion, cells were further purified from the interphase of 37.5% and 75% Percoll gradient after 20 min spin at 2,500 rpm at room temperature. For flow cytometry analysis, cells were stained using Live and Dead violet viability kit (Invitrogen) or Zombie Aqua fixable viability kit (BioLegend). CD16/32 antibody (Thermo Fisher) was used to block the nonspecific binding followed by surface molecule staining on ice for 30 min. Cells were fixed and permeabilized with Foxp3 staining buffer Kit (eBioscience) for transcription factor staining. For cytokine staining, cells were stimulated with 50 ng/mL PMA and 500 ng/mL ionomycin for 3 h and Brefeldin A (2 μg/mL) was added 2 h before cells were collected. Sample acquisition was performed on BD FACSCantoII or Cytek Aurora flow cytometer and analyzed with FlowJo software (version 10.2).
Cd16 32 antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CD16/32 antibody is a laboratory reagent used in flow cytometry and other immunological assays. It binds to the Fc gamma receptor III and II (CD16 and CD32), which are expressed on the surface of various immune cells, such as natural killer cells, macrophages, and granulocytes. This antibody can be used to identify and characterize these cell types in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (4 mentions)
Live and dead violet viability kit, by Thermo Fisher Scientific (2 mentions)
Foxp3 staining buffer kit, by Thermo Fisher Scientific (2 mentions)
Zombie aqua fixable viability kit, by BioLegend (2 mentions)
70 m nylon mesh, by BD (2 mentions)
Liberase, by Roche (2 mentions)
Accuri c6, by BD (2 mentions)
Efluor 780, by Thermo Fisher Scientific (2 mentions)
Facsdiva software, by BD (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
F4 80, by Bio-Rad (1 mentions)
Dulbecco s modified eagle medium, by Roche (1 mentions)
Cd62l, by Thermo Fisher Scientific (1 mentions)
Cxcr3, by BioLegend (1 mentions)
Siglec f, by BD (1 mentions)
Cd40l, by BioLegend (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Rock inhibitor y 27632, by STEMCELL (1 mentions)
Facsaria 3, by BD (1 mentions)
Lsrii fortessa, by BD (1 mentions)
18 protocols using cd16 32 antibody
1
Isolation and Characterization of Lymphocytes
2
Isolation and Characterization of Lymphocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The isolation of lymphocytes from spleen, peripheral/mesenteric lymph nodes, small and large intestinal lamina propria, and small intestine intraepithelial lymphocytes were performed as previously described.71 (link) After digestion, cells were further purified from the interphase of 37.5% and 75% Percoll gradient after 20 min spin at 2,500 rpm at room temperature. For flow cytometry analysis, cells were stained using Live and Dead violet viability kit (Invitrogen) or Zombie Aqua fixable viability kit (BioLegend). CD16/32 antibody (Thermo Fisher) was used to block the nonspecific binding followed by surface molecule staining on ice for 30 min. Cells were fixed and permeabilized with Foxp3 staining buffer Kit (eBioscience) for transcription factor staining. For cytokine staining, cells were stimulated with 50 ng/mL PMA and 500 ng/mL ionomycin for 3 h and Brefeldin A (2 μg/mL) was added 2 h before cells were collected. Sample acquisition was performed on BD FACSCantoII or Cytek Aurora flow cytometer and analyzed with FlowJo software (version 10.2).
3
Multiparameter Flow Cytometry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Non-adherent cells collected from co-cultures at each time point were stained with antibodies specific for myeloid and dendritic-like cells. As described previously [16 (link), 24 (link), 25 (link)], 105–6 cells were incubated with CD16/32 antibody (Clone 93; eBioscience) for 15 min to block surface Fc receptors. Cells were then washed with DMEM/1%FCS/0.1%NaN3 (FACS buffer) and stained with primary antibodies specific for CD8α, B220, CD115, F4/80, 4-1BBL, CD11c, CD11b, MHC-II and Sirp-α for 20 min on ice. Secondary fluorochrome conjugates were added after a washing step and incubated for a further 20 min on ice. Cells were then washed twice and resuspended in FACS buffer for flow cytometric analysis. Cells were stained with propidium iodide (PI) (1 μg/ml) for live cell discrimination in order to gate PI− live cells. FSC versus SSC plots were used to gate large cells for marker analysis. Cell acquisition involved use of a LSR II flow cytometer (Becton Dickinson), and between 5 × 104 and 1 × 106 events were collected for each sample. Gates were set to delineate cell subsets using isotype control antibodies and ‘fluorescence minus one’ (FMO) controls. Numbers on gates reflect % positive cells. Cell subset analysis was performed using BD FACSDiva Software (Becton Dickinson) and FlowJo Software (Tristar; Phoenix, Arizona, USA).
4
Comprehensive Immune Profiling of Adipose Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
White blood cells from blood, spleen, lymph nodes (LN), liver and stromal vascular fraction (SVF) from EpAT were analyzed by flow cytometry. EpAT was minced into small pieces and digested with liberase (0.25 mg/mL, Roche) for 45 minutes at 37 °C. The digested samples were passed through a 70-µm nylon mesh (BD Biosciences, Breda, the Netherlands). The SVF was obtained from the resulting pellet and resuspended in FACS buffer. Erythrocytes in blood and spleen were removed by incubation with hypotonic lysis buffer (8.4 g of NH4Cl and 0.84 g of NaHCO3 per liter of distilled water). To prevent non-specific binding of antibodies to the Fc receptor, all cell suspensions were incubated with CD16/32 antibody (eBioscience, San Diego, CA, USA) in FACS buffer (0.5% BSA, 0.01% NaN3 in PBS) before the antibodies were incubated with the indicated tissues. Fluorescence was measured by flow cytometry (FACSCanto II, BD Biosciences, Breda, The Netherlands) and analyzed with FlowJo software version 7.6.5. (Tree star). The antibodies used are listed in Supplemental Table 1 .
5
Multiparametric Flow Cytometry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For flow cytometric analysis, blood was obtained by cardiac puncture with an EDTA-coated syringe. Spleen and LNs were removed and collected in phosphate-buffered saline (PBS). EpAT and ScAT were prepared separately by mincing into small pieces and digested using liberase (0.25 mg/mL Dulbecco’s Modified Eagle Medium, Roche) for 45 min at 37°C. Spleen, LN, and digested adipose tissue samples were passed through a 70 µm nylon mesh (BD Biosciences). The stromal vascular fraction (SVF) was collected by pelleting of the suspension and diluted in fluorescence-activated cell sorting (FACS) buffer (0.5% bovine serum albumin, 0.01% NaN3 and 2 mM EDTA in PBS). Blood and spleen were incubated with hypotonic lysis buffer (8.4 g NH4Cl and 0.84 g NaHCO3 in 1 L miliQ) to remove erythrocytes. Cell suspensions were blocked with CD16/32 antibody (eBioscience, Waltham, Massachusetts, USA) in FACS buffer to prevent non-specific binding. Indicated tissues were incubated with CD45, CD19, CD62L, CD44, FoxP3, Ly6C (eBioscience), CD3, CD8, CD38, CXCR3, CD40L (Biolegend, San Diego, California, USA), CD4, CD11b, Ly6G, SiglecF (BD Biosciences), and F4/80 (AbD serotec) antibodies. Fluorescence was measured by flow cytometry (FACSCanto II, BD Biosciences) and analysed with FlowJo software V.10 (Tree star).
6
Prostate Single-Cell Suspension Immunophenotyping
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse prostate single cell suspension was blocked with the Fc blocker (CD16/32 antibody from eBioscience) for 40 min at room temperature. Staining antibodies were diluted in 4% FBS buffer containing Y-27632 ROCK inhibitor (STEMCELL Technologies, #72302) and applied to the prostate single cell suspension for 40 min at room temperature. Flow cytometry analysis or sorting was conducted using BD Accuri C6 or FACSAria III flow cytometer. Antibodies used in the study are listed in Supplementary Table 2 . Propidium Iodide (PI, Invitrogen) was added to sample before sorting to gate viable cells.
7
Multiplex Flow Cytometry of Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Blood was obtained by cardiac puncture and collected using EDTA-filled syringes. The spleen and lymph nodes were collected. Erythrocytes in the blood and spleen were removed by incubation with hypotonic lysis buffer (8.4 g of NH4Cl and 0.84 g of NaHCO3 per litre of distilled water). To prevent non-specific binding of antibodies to the Fc receptor, all cell suspensions were incubated with a CD16/32 antibody (eBioscience, San Diego, CA, USA) prior to labelling. CD45, CD19, CD8, FoxP3 (eBioscience, San Diego, CA, USA), CD3 (BioLegend, San Diego, CA, USA), CD11b, and CD4 (BD, Breda, The Netherlands) antibodies were incubated with the indicated tissues. Staining was analysed by flow cytometry (FACSCanto II, BD Biosciences, Breda, The Netherlands) and FlowJo software version 7.6.5 (Tree Star).
8
FACS Analysis of Tumor-Derived Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For FACS analysis primary tumors or spleens were collected and maintained in DMEM−10%FBS, then minced and filtered through a 40 μm-pores cell strainer (BD). Red blood cells were removed using ACK lysis buffer (ammonium chloride potassium). Cells were Fc-blocked using CD16/32 antibody (eBioscience) before staining. Antibodies used were: CD45.2; Gr-1; CD11b; Ly6G, and Ly6C (all from eBioscience). Samples were acquired using a BD LSR II Fortessa instrument and analyzed with FlowJo software (TreeStar). All samples are analyzed in single; in each experiment at least 3–4 samples were analyzed for each group.
9
Comprehensive Immune Cell Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single cells were isolated from collected spleens by centrifugation in a gradient lymphocyte isolation solution for mice. Next, single-cell suspensions were blocked with CD16/32 antibody (14-0161-85; eBioscience) and then stained with antibodies against mouse CD4 (fluorescein isothiocyanate [FITC], 100509; BioLegend), CD8 (phycoerythrin [PE] or FITC, 100707 or 100705; BioLegend), CD3 (FITC, 100305; BioLegend), NK-1.1 (PE/Cy5, 108715; BioLegend), CD44 (allophycocyanin [APC], 103011; BioLegend), CD45 (PerCP/Cy5.5, 103132; BioLegend), CD11b (PE, 101207; BioLegend), CD11c (FITC, 557400; BD Biosciences), Ly-6G (APC, 127613; BioLegend), CD278 (PE, 117405; BioLegend), CD25 (APC, 17-0251-81; eBioscience), F4/80 (FITC, 11-4801-81; eBioscience), Gr-1 (FITC, 108405; BioLegend), TIGIT (PE/Cy7, 142107; BioLegend), CD152 (PerCP/Cy5.5, 106315; BioLegend), CD366 (PerCP/Cy5.5, 134011; BioLegend), or CD223 (PerCP/Cy5.5, 125211; BioLegend), or intracellularly stained with IL-2 (APC, 503809; BioLegend), IFN-γ (APC, 17-7311-81; eBioscience), TNF-α (APC, 17-7321-81; eBioscience), Ki-67 (PE, 652404; BioLegend), or Foxp3 (PE, 12-4771-80; eBioscience) following the instructions of the Foxp3/Transcription Factor Staining Buffer Set (00-5523-00; eBioscience). Samples were collected on BD LSR II, and data were analyzed using FlowJo software.
10
Microglial and Astrocytic Cell Isolation
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!