BMDMs were harvested from culture plates using 4 mmol/L EDTA in PBS for 10 minutes at 37°C. Cells were washed in flow cytometry buffer (PBS with 2% fetal bovine serum and 1 mmol/L EDTA) and stained using the following antibodies (all BD Bioscience unless stated): F4/80 (BM8; e-bioscience), CD11c (HL3), CD206 (C068C2; Biolegend), CD69 (H1.2F3), and CD40 (3/23). Fc block (4.4G2) was included in all stains. Data were acquired on a LSR II flow cytometer (Becton Dickinson) and analyzed using FlowJo software (TreeStar). BMDM culture supernatants were collected after 24-hour treatment with the differentiation or activation conditions. Levels of cytokines were quantified by standard sandwich ELISA using paired antibody kits (e-bioscience).
Paired antibody kits
Manufactured by Thermo Fisher Scientific
Paired antibody kits are laboratory tools used to detect and quantify specific proteins or molecules in a sample. These kits contain two antibodies, one that binds to the target analyte and another that is used for detection. The kits provide a standardized and reliable method for conducting immunoassays.
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3 protocols using paired antibody kits
1
Flow Cytometric Analysis of Bone Marrow-Derived Macrophages
2
Antigen-Specific T Cell Activation Assay
3
Antigen-Specific T Cell Activation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
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DCs were activated with 100ng/ml LPS (Sigma-Aldrich, MO, USA) overnight and loaded with 1μM ovalbumin peptide 323-339 (AnaSpec, CA, USA) for 90min. After washing, DCs were cultured with purified CD4 OT-II T cells at a ratio of 1:10.
Moloney murine leukaemia virus H19env peptide 123-141 was emulsified in Complete Freund’s Adjuvant (CFA, Sigma-Aldrich, MO, USA) using a sonicator. Mice were immunized with a total of 100μg peptide/CFA subcutaneously into the hind legs. Alternatively, DCs were activated with 100ng/ml LPS (Sigma-Aldrich, MO, USA) overnight and loaded with 50μg/ml H19env peptide for 90min. A total of 1-2×106 LPS-activated peptide-loaded DCs were injected intravenously into mice. DCs were loaded with 25μg/ml Schistosomal egg antigen (SEA, in-house) or 10μg/ml heat-killed Propionibacterium acnes (in-house) overnight. A total of 5×105 cells were injected into the feet of recipient C57Bl/6 mice. 7d post-immunization, popliteal (draining) lymph nodes were extracted, and cells restimulated with antigens (15μg/ml SEA, 1μg/ml heat-killed P. acnes, plate-bound anti-CD3 (clone 2C11)) in vitro for 72hr. Supernatants were taken and cytokine production analyzed by ELISA using paired antibody kits (ebioscience) according to standard protocols.
Moloney murine leukaemia virus H19env peptide 123-141 was emulsified in Complete Freund’s Adjuvant (CFA, Sigma-Aldrich, MO, USA) using a sonicator. Mice were immunized with a total of 100μg peptide/CFA subcutaneously into the hind legs. Alternatively, DCs were activated with 100ng/ml LPS (Sigma-Aldrich, MO, USA) overnight and loaded with 50μg/ml H19env peptide for 90min. A total of 1-2×106 LPS-activated peptide-loaded DCs were injected intravenously into mice. DCs were loaded with 25μg/ml Schistosomal egg antigen (SEA, in-house) or 10μg/ml heat-killed Propionibacterium acnes (in-house) overnight. A total of 5×105 cells were injected into the feet of recipient C57Bl/6 mice. 7d post-immunization, popliteal (draining) lymph nodes were extracted, and cells restimulated with antigens (15μg/ml SEA, 1μg/ml heat-killed P. acnes, plate-bound anti-CD3 (clone 2C11)) in vitro for 72hr. Supernatants were taken and cytokine production analyzed by ELISA using paired antibody kits (ebioscience) according to standard protocols.
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