Log In Sign up
The largest database of trusted experimental protocols

Blood dendritic cell enumeration kit

Manufactured by Miltenyi Biotec
Visit Supplier
Sourced in United Kingdom

The Blood Dendritic Cell Enumeration Kit is a laboratory tool used to quantify dendritic cells in human blood samples. It provides a standardized method to enumerate the different subtypes of dendritic cells present in the sample.

Automatically generated - may contain errors

Lab products found in correlation

Facscanto 2, by BD (2 mentions) Tnf α, by Merck Group (1 mentions) Lipopolysaccharides (lps), by Merck Group (1 mentions) Tnf α, by R&D Systems (1 mentions) Flow count fluorosphere, by Beckman Coulter (1 mentions) Fixation permeabilization buffer set, by Thermo Fisher Scientific (1 mentions) Monensin, by Thermo Fisher Scientific (1 mentions) Efluor 780, by Thermo Fisher Scientific (1 mentions) Ionomycin, by Merck Group (1 mentions) Phorbol myristate acetate (pma), by Merck Group (1 mentions) Lymphoprep, by Axis-Shield (1 mentions) Fcr block, by Miltenyi Biotec (1 mentions) Cd14 pe cy5, by Thermo Fisher Scientific (1 mentions) Facscalibur, by BD (1 mentions)

5 protocols using blood dendritic cell enumeration kit

1

Dendritic Cell Activation Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols
RBcs was prepared by seeding 10-cm dish (Falcon; BD Bioscience, Franklin Lakes, NJ, USA) with 1 × 107 RB cell in 10 mL of completed medium for 24 h and centrifuged to remove cell debris. On day 5 of DC culture, RBcs was added to test DCs, while the same volume of culture medium was added to control DCs. On day 6, maturation of DCs was induced by adding 20 ng/mL of TNF-α (R&D System) or 1 μg/mL of LPS (Sigma), and the phenotype of DCs was determined by flow cytometry after incubating with TNF-α or LPS for 24 h. In some experiments, before adding of TNF-α or LPS, DCs were purified on day 6. Briefly, RBcs-treated DCs or control DCs were washed extensively and then purified with microbeads on auto-MACS columns using a Blood Dendritic Cell Isolation kit (Miltenyi Biotech, BergischGladbach, Germany) according to the manufacturer’s instructions. In short, isolation of DCs was performed in a two-step procedure. First, cells labeled with the Non-DC Depletion Cocktail comprising with CD14 and CD19 magnetic beads were depleted. Then DCs were positively selected by labeled with DC Enrichment Cocktail comprising with CD1c (BDCA-1), CD304 (BDCA-4/Neuropilin-1), and CD141 (BDCA-3) magnetic beads. Purified DCs samples contained >95% CD1c+ DCs as evaluated by the Blood Dendritic Cell Enumeration Kit (Miltenyi Biotech).
Ma J., Han H., Ma L., Liu C., Xue X., Ma P., Li X, & Tao H. (2014). The immunostimulatory effects of retinoblastoma cell supernatant on dendritic cells. Protein & Cell, 5(4), 307-316.
+ Open protocol
+ Expand
2

Enumeration of Blood Dendritic Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
DCs were enumerated in heparinized blood using a Blood Dendritic Cell Enumeration Kit (Miltenyi Biotec Ltd., Surrey, United Kingdom) according to the manufacturer’s instructions. The anti-BDCA cocktail contains: anti CD303 (BDCA-2) Fluorescein (FITC), anti-CD1c (BDCA-1) PE, anti-CD14 Phycoerythrin-Cyanine 5 (PE-Cy5), and anti-CD19 PE-Cy5 for the determination of plasmacytoid (pDC) and myeloid (mDC) DC. Antibodies to CD14 and CD19 antigens were used for exclusion of monocytes and B cells, respectively. To enable quantification in terms of absolute counts/μL blood, Flow count fluorospheres (Beckman Coulter) were added to each sample prior to flow cytometric (EC800 Sony) data acquisition. Analysis was performed using FlowJo software (FlowJo, United States). The ratio of mDC to pDC were calculated by applying the following formula DC = number of DC/number count beads × concentration).
Maijo M., Ivory K., Clements S.J., Dainty J.R., Jennings A., Gillings R., Fairweather-Tait S., Gulisano M., Santoro A., Franceschi C., Carding S.R, & Nicoletti C. (2018). One-Year Consumption of a Mediterranean-Like Dietary Pattern With Vitamin D3 Supplements Induced Small Scale but Extensive Changes of Immune Cell Phenotype, Co-receptor Expression and Innate Immune Responses in Healthy Elderly Subjects: Results From the United Kingdom Arm of the NU-AGE Trial. Frontiers in Physiology, 9, 997.
+ Open protocol
+ Expand
3

Multiparametric FACS analysis of PBMCs

Check if the same lab product or an alternative is used in the 5 most similar protocols
PBMC were isolated by density gradient centrifugation (Lymphoprep; Axis Shield, Oslo, Norway). 1 × 106 PBMC were incubated with fixable viability dye eFluor®780 (eBioscience), FcR block (Miltenyi), and the respective fluorochrome‐conjugated antibodies: αCD3‐eFluor450 (UCHT1, eBioscience), αCD4‐eFluor450 (SK3, Biolegend), αCD8‐APC (SK1, BD Biosciences), αCD19‐PE/Cy7 (HIB19, Biolegend), αCD56‐PE (HCD56, Biolegend), αFoxp3‐PE (206D, Biolegend), αIFNγ‐APC (B27, BD Biosciences), αIL‐4‐FITC (MP4‐25D2, Biolegend), αIL‐17A‐PE (eBio64DEC17, eBioscience), and analyzed on a BD FACSCantoII using FACSDiva software. For intracellular cytokine staining, cells were prestimulated for 3h with ionomycin (1 μmol/L, Sigma‐Aldrich) and PMA (50 ng/mL, Sigma‐Aldrich) in the presence of monensin (2 μmol/L, eBioscience), fixed and made permeable with the Fixation/Permeabilization buffer set (eBioscience) according to the manufacturer's protocol.
Peripheral blood DC were analyzed for the expression of CD1c (BDCA‐1), CD303 (BDCA‐2), and CD141 (BDCA‐3) with the Blood Dendritic Cell Enumeration Kit (Miltenyi) in a subset of patients (frozen samples) according to the manufacturer′s instructions.
Hammer A., Waschbisch A., Kuhbandner K., Bayas A., Lee D., Duscha A., Haghikia A., Gold R, & Linker R.A. (2018). The NRF2 pathway as potential biomarker for dimethyl fumarate treatment in multiple sclerosis. Annals of Clinical and Translational Neurology, 5(6), 668-676.
+ Open protocol
+ Expand
4

Dendritic Cell Subset Identification

Check if the same lab product or an alternative is used in the 5 most similar protocols
Dendritic cells were isolated from PBMC using the Blood Dendritic Cell Enumeration kit (Miltenyl Biotec). Dendritic cell subsets were identified by anti-BDCA-2+ (CD11c−/CD123+high) for pDCs, anti-BDCA-1+ (CD11c+ high/CD123+ low) for mDC1 and anti-BDCA-3+ (CD11c+low/CD123−) for mDC2(Dzionek et al., 2000 ). Samples were also labeled with CD19-PE-Cy2 and CD14-PE-Cy5 (eBiosciences), for exclusion of B cells and monocytes. Cells were analyzed by FACSCalibur (BD Biosciences).
Kitcharoensakkul M., Bacharier L.B., Yin-Declue H., Boomer J.S., Burgdorf D., Wilson B., Schechtman K, & Castro M. (2016). Temporal Biological variability in dendritic cells and regulatory T cells in peripheral blood of healthy adults. Journal of immunological methods, 431, 63-65.
+ Open protocol
+ Expand
5

Multicolor Flow Cytometry for Dendritic Cell Subsets

Check if the same lab product or an alternative is used in the 5 most similar protocols
Dendritic cell analysis was performed with the Blood Dendritic cell enumeration kit (#130-091-086; Miltenyi Biotec). This assay is based on dendritic cell–specific surface antigens CD303 (BDCA-2), CD141 (BDCA-3), and CD1c (BDCA-1). Three distinct dendritic cell subsets were identified in whole blood: plasmacytoid dendritic cells as CD303+ (FITC clone: AC144, BDCA-2), type 1 myeloid dendritic cells (MDC1s) as CD1c+ (PE clone: AD5-8E7, BDCA-1), and type 2 myeloid dendritic cells (MDC2s) as CD141+ (APC clone: Ad4-14H12, BDCA-3) surface expression. Gate exclusion for CD19, CD14, and dead cells was performed. After 10 min of incubation in ice with the mix of antibodies, red blood cells were lysed. Cells were washed with PBS, fixed with 1% PFA for 10 min, acquired with a FACSCanto II (Becton Dickinson), and analyzed with FlowJo software (Tree Star Inc.; version 8.8.6). At least 106 total events were acquired.
Lam M.T., Coppola S., Krumbach O.H., Prencipe G., Insalaco A., Cifaldi C., Brigida I., Zara E., Scala S., Di Cesare S., Martinelli S., Di Rocco M., Pascarella A., Niceta M., Pantaleoni F., Ciolfi A., Netter P., Carisey A.F., Diehl M., Akbarzadeh M., Conti F., Merli P., Pastore A., Levi Mortera S., Camerini S., Farina L., Buchholzer M., Pannone L., Cao T.N., Coban-Akdemir Z.H., Jhangiani S.N., Muzny D.M., Gibbs R.A., Basso-Ricci L., Chiriaco M., Dvorsky R., Putignani L., Carsetti R., Janning P., Stray-Pedersen A., Erichsen H.C., Horne A., Bryceson Y.T., Torralba-Raga L., Ramme K., Rosti V., Bracaglia C., Messia V., Palma P., Finocchi A., Locatelli F., Chinn I.K., Lupski J.R., Mace E.M., Cancrini C., Aiuti A., Ahmadian M.R., Orange J.S., De Benedetti F, & Tartaglia M. (2019). A novel disorder involving dyshematopoiesis, inflammation, and HLH due to aberrant CDC42 function. The Journal of Experimental Medicine, 216(12), 2778-2799.
+ 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?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!