Isolation and Differentiation of Bovine Dendritic Cells

Blood was collected from healthy cattle in 60 ml syringes containing 7.5% EDTA. PBMCs were obtained from these blood samples using the Ficoll (GE Healthcare Bioscience) method of isolation.²¹ CD14⁺ monocytes were isolated from PBMCs by positive selection using MACs columns (Miltenyi Biotec Inc.). Mature bDCs were subsequently derived from CD14⁺ monocytes after culturing in a complete Roswell Park Memorial Institute-1640 (RPMI-1640) medium supplemented with 10% heat-inactivated FBS and 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) (Hyclone Laboratories Inc.), 2-mercaptoethanol (Sigma-Aldrich), recombinant bovine GM-CSF at 100 ng/ml (Bio-Rad) and IL-4 at 50 ng/ml (Biorad) as previously described.^{22 (link)} bDCs were assessed by flow cytometry and the expression of DC-specific marker CD206 was found to be greater than 97%.

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Ihedioha O., Potter A.A, & Chen J.M. (2020). Poor stimulation of bovine dendritic cells by Mycobacterium bovis culture supernatant and surface extract is associated with decreased activation of ERK and NF-κB and higher expression of SOCS1 and 3. Innate Immunity, 26(6), 537-546.

Publication 2020

Ficoll MACs columns 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) 2-mercaptoethanol IL-4

2 mercaptoethanol Acid Bdcs Blood Bovine Cattle Culturing medium Edta Ficoll Flow cytometry Gm csf Hepes Isolation Monocytes Syringes

Corresponding Organization : University of Saskatchewan

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Variable analysis

independent variables

Isolation of CD14+ monocytes from PBMCs using MACS columns
Culturing CD14+ monocytes in RPMI-1640 medium supplemented with 10% FBS, HEPES, 2-mercaptoethanol, recombinant bovine GM-CSF (100 ng/ml), and IL-4 (50 ng/ml)

dependent variables

Maturation of CD14+ monocytes into bDCs
Expression of DC-specific marker CD206 on bDCs (>97%)

control variables

Blood collected from healthy cattle
Blood collected in 60 ml syringes containing 7.5% EDTA
PBMCs isolated from blood samples using Ficoll method

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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