Cd14 pedazzle594

Manufactured by BioLegend

Sourced in United States

The CD14-PEDazzle594 is a fluorescently labeled antibody product from BioLegend. It is designed to detect the CD14 cell surface marker in flow cytometry and other immunoassay applications.

Automatically generated - may contain errors

Lab products found in correlation

5 protocols using cd14 pedazzle594

Comprehensive Immunophenotyping of PBMCs

Check if the same lab product or an alternative is used in the 5 most similar protocols

PBMCs were stained with the Zombie Aqua Flexibility Viability dye (BioLegend, San Diego, CA, USA) for the live/dead gate for 15 min at room temperature, followed by the antibodies for (1) TLR stimulation experiments: CD3-PE-Cy7, CD19-APC-H7, CD56-BV421, CD16-BUV395, HLA-DR-BB515, CD11b-BV605, CD11c-APC (all from Becton Dickinson, Franklin Lakes, NJ, USA) and CD14-PEDazzle594 (BioLegend); and (2) time-course experiments: a lineage-negative (lin⁻) cocktail (CD3/CD19/CD20/CD56/CD14/CD16-AF700, Bio-Rad, Hercules, CA, USA), CD14-PEDazzle594, HLA-DR⁺-BB515 (Biolegend), CD123-PE (BioLegend), CD11b-BV605 (Becton Dickinson), CD11c-APC (Becton Dickinson), CD141-BV711 (Becton Dickinson), CD1c-BV421 (BioLegend) and CD1aPerCP/Cy5.5 (Biolegend) for 20 min on ice, washed twice and resuspended in flow cytometry buffer (phosphate-buffered saline (PBS) + 2% fetal bovine serum (FBS)).
All samples were run on an LSR Fortessa X-20 (Becton Dickinson) with a minimum of 100,000 events recorded per sample. All data were analyzed with FlowJo Software v10.7.1 (Ashland, OR, USA).

Mazarakis N., Anderson J., Toh Z.Q., Higgins R.A., Do L.A., Luwor R.B., Snibson K.J., Karagiannis T.C, & Licciardi P.V. (2021). Examination of Novel Immunomodulatory Effects of L-Sulforaphane. Nutrients, 13(2), 602.

+ Open protocol

+ Expand

Flow Cytometry Sample Preparation

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were transferred from the 96 well plate to a PCR plate (Thermo Fisher) and centrifuged at 400 x g for 5 min at room temperature, making sure to aspirate the resulting supernatant in between each transfer. Once all cells were transferred to the new PCR plate, cells were centrifuged and resuspended in FBS Stain Buffer (BD Biosciences). FC Block Cocktail (Human TruStain FcX Fc Receptor Blocking Solution [Biolegend] and FBS Stain Buffer) was added to each sample and incubated for 10 min at 21 °C. Following the blocking incubation, Live Stain Cocktail, composed of Live/Dead-Aqua (1:100, Biolegend), CD8-PerCP-Cy5.5 (1:20, Biolegend), and CD14-PE-Dazzle594 (1:20, Biolegend) in FBS Stain Buffer, was added to each well, and the plate was incubated for a subsequent 30 min at 21 °C. Samples were washed twice via centrifugation and resuspension in 150 μL of FBS Stain Buffer, and finally resuspended in 60 μL of FBS Stain Buffer. 16% PFA was added to each sample for a final concentration of 1.6% PFA and incubated for 10 min at 21 °C to fix the cells. After fixation, cells were washed as before and resuspended in FBS Stain Buffer in preparation for intracellular staining.

Chen H., Schürch C.M., Noble K., Kim K., Krutzik P.O., O’Donnell E., Vander Tuig J., Nolan G.P, & McIlwain D.R. (2020). Functional comparison of PBMCs isolated by Cell Preparation Tubes (CPT) vs. Lymphoprep Tubes. BMC Immunology, 21, 15.

+ Open protocol

+ Expand

Analyzing MAIT Cell Activation by Flow Cytometry

Check if the same lab product or an alternative is used in the 5 most similar protocols

All cellular events were analyzed via flow cytometry on a BD Fortessa running FACSDIVA v6.1 (BD, San Jose, CA, USA) software. Our gating strategy has been described elsewhere (24 (link)), with Vα7.2 and CD161 cells being used to identified MAIT cells from the CD3⁺ population with gating on CD8⁺ and CD4⁺ to identify specific subpopulations. Additional markers were used to determine the activation status (CD69), homing marker expression (CCR4, CCR5, and CCR6), or intracellular cytokines of MAIT cells with exercise, along with fluorescence minus one (FMO) controls. CD14 (PE-Dazzle594; Biolegend) and CD66 (APC, Novus Biologicals, LLC, Littleton, CO, USA) were used initially to confirm the desired lymphocyte population did not consist of monocytes or neutrophils. Unlabeled, single color compensation and AbC™ Total Antibody Compensation Bead Kit (Thermo Fisher Scientific, Hampton, NH, USA) for markers with low expression (i.e. CD69, CCR4, CCR5, CCR6, IL-17, TNFα, and IFNγ) were used for every experiment in addition to fluorescence minus one (FMO) controls for PE as this channel was used to detect Vα7.2. Gating analyses were completed using FlowJo version 10.1r7 (Ashland, OR, USA). Circulating cell number was determined by multiplying the percentage of lymphocytes expressing the markers of interest with the hematology total lymphocyte count for each specific cell population.

Hanson E.D., Danson E., Evans W.S., Wood W.A., Battaglini C.L, & Sakkal S. (2019). Exercise Increases MAIT Cell Cytokine Expression but not Activation or Homing Markers. Medicine and science in sports and exercise, 51(2), 379-388.

+ Open protocol

+ Expand

Phenotypic Analysis of Cryopreserved PBMCs

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cryopreserved PBMCs were thawed and washed twice with 10 mL of FACS buffer (1 x PBS containing 2% FBS and 1 mM EDTA) and resuspended in 100 uL of 1x PBS containing Zombie UV live/dead dye at 1:200 dilution (BioLegend, 423108) and incubate at room temperature for 15 minutes. Following washing, cells were incubated with an antibody cocktail for 1 hour protected from light on ice. The following antibodies were used: IgD PE (BD Biosciences, 555779), IgM PerCP-Cy5.5 (BioLegend, 314512), CD20 APC-H7 (BD Biosciences, 560734), CD27 PE-Cy7 (BioLegend, 302838), CD14 PE/Dazzle^™ 594 (BioLegend, 301852), CD16 BV605 (BioLegend, 302040), IgG BV650 (BD Biosciences, 740596), CD3 BUV737 (BD Biosciences, 612750) and Alexa Fluor 488-labeled Wuhan spike (SinoBiological, 40589-V27B-B), and BV421 labeled Omicron Spike (SinoBiological^™, 40589-V49H3-B). All antibodies were used as the manufacturer’s instruction and the final concentration of each probe was 0.1 ug/ml. Cells were washed twice in FACS buffer and immediately acquired on a BD FACS Aria III for acquisition and FlowJo for analysis.

Wimmers F., Burrell A.R., Feng Y., Zheng H., Arunachalam P.S., Hu M., Spranger S., Nyhoff L., Joshi D., Trisal M., Awasthi M., Bellusci L., Ashraf U., Kowli S., Konvinse K.C., Yang E., Blanco M., Pellegrini K., Tharp G., Hagan T., Chinthrajah R.S., Grifoni A., Sette A., Nadeau K.C., Haslam D.B., Bosinger S.E., Wrammert J., Maecker H.T., Utz P.J., Wang T.T., Khurana S., Khatri P., Staat M.A, & Pulendran B. (2023). Systems biological assessment of the temporal dynamics of immunity to a viral infection in the first weeks and months of life. medRxiv.

+ Open protocol

+ Expand

Immunophenotyping Murine Immune Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Mice were euthanised with CO₂, and blood was collected by cardiac puncture into EDTA-containing tubes (100 mmol/l EDTA). After lysing erythrocytes with RBC lysis buffer (eBiosciences, San Diego, USA) and washing with PBS, antibodies and viability dye were incubated with white blood cells in FACS buffer (PBS with 0.5% BSA and 2 mmol/l EDTA) for 20 min at 4°C. The following antibodies were used (Biolegend, San Diego, USA): CD45-FITC (30-F11 clone), Ly6G-Brilliant Violet 510 (1A8 clone), Ly6C-PE-Cy7 (HK1.4 clone), CD11b-APC (M1/70 clone), F4/80-PE (BM8 clone), CCR2-Brilliant Violet 421 (SA203G11 clone) and CD14-PE-Dazzle 594 (Sa14-2 clone). Viability dye (Fixable Viability Dye eFluor 780, eBiosciences) was added to distinguish live cells. Cells were washed in FACS buffer and fixed for 20 min at 4°C (Perm/Fix buffer, BD Biosciences, USA) before analysis on a Novocyte cytometer (Acea, USA).

Saadane A., Veenstra A.A., Minns M.S., Tang J., Du Y., Abubakr Elghazali F., Lessieur E.M., Pearlman E, & Kern T.S. (2023). CCR2-positive monocytes contribute to the pathogenesis of early diabetic retinopathy in mice. Diabetologia, 66(3), 590-602.

+ Open protocol

+ Expand

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?

Revolutionizing how scientists
search and build protocols!