GIMAP6fl/flCD2Cre mice or control GIMAP6fl/fl mice were immunised with 4-hydroxy-3-nitrophenylacetyl (NP)19–keyhole limpet hemocyanin (KLH; Biosearch Technologies, Novato, CA) adsorbed to alum as described previously [36 ]. Mice were bled at the indicated times, before boosting with soluble antigen and antibody titres determined by ELISA essentially as previously described [63 ] except that NP23-BSA was replaced by NP20-BSA.
Keyhole limpet hemocyanin klh
Manufactured by Biosearch Technologies
Sourced in United States
Keyhole limpet hemocyanin (KLH) is a large, copper-containing respiratory protein found in the hemolymph of the marine mollusk Megathura crenulata, commonly known as the keyhole limpet. KLH serves as a carrier protein and has been widely used in various immunological applications.
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3 protocols using keyhole limpet hemocyanin klh
1
NP-KLH Immunization Assay in Mice
2
Optimizing Dendritic Cell and Splenocyte Culture Conditions
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The cell culture medium for DCs and splenocytes was RPMI 1640 (Gibco-BRL, Grand Island, NY, USA) supplemented with 10% fetal calf serum (FCS; Gibco-BRL) and 100 U/mL streptomycin-penicillin. Granulocyte-macrophage colony stimulating factor (GM-CSF; Lot no. 050755-1), IL-4 (Lot no.1106CY49), and IL-2 (Lot no.0608108) were obtained from Peprotech (Rocky Hill, NJ, USA). PC-keyhole limpet hemocyanin (PC-KLH) and keyhole limpet hemocyanin (KLH) were obtained from Biosearch Technologies (Novato, CA, USA). The purified anti-CD4 antibody (Ab; clone RM4-5) was from BD Systems (Franklin, NJ, USA). Fluorescein isothiocyanate (FITC)-conjugated anti-CD11c (Clone N418), phycoerythrin (PE)-conjugated anti-CD86 (Clone PO3.1), anti-major histocompatibility complex Class II (MHC-II)-APC (Clone HIS19), anti-CD40-PE (Clone 5C3), anti-CD45-FITC (Clone HI30), anti-CD19-APC (Clone HIB19), anti-CD4-FITC (Clone GK1.5), anti-CD25-APC (Clone PC61.5), anti-Foxp3-PE (Clone FJK-165), anti-interferon (IFN)-γ-PE (Clone XMG1.2), anti-IL-17A-PE (Clone eBio17B7), anti-Foxp3-biotin (Clone FJK-16 s), and anti-CD11c-biotin (Clone N418) Abs were obtained from eBioscience (San Diego, CA, USA). Anti-CD68 Ab was obtained from Millipore and anti-smooth muscle actin (SMA) Ab (Lot no. BM0002) was obtained from Boster (Wuhan, China). Control rat IgG was obtained from ThermoFisher Scientific (Rockford, IL, USA).
3
KLH-induced Delayed-type Hypersensitivity
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On day 7, each rat was subcutaneously injected with 0.1 ml of normal saline containing 50 μg of Keyhole Limpet Hemocyanin (KLH) (Biosearch Technologies, Inc., USA) on the right hind footpad and the left hind footpad received 0.1 ml of normal saline only. On day 14, the right hind footpad received 0.1 ml of normal saline only (i.e. control foot). The left hind footpad was challenged subcutaneously with 0.1 ml of normal saline containing 50 μg of KLH. The thickness of the left and right hind footpad was measured 24 hours later using RD DC 706 digital Vernier calipers (Raider Pro professional Tools Co. Ltd., China). The difference in the thickness of the right hind footpad and the left hind footpad was used as a measure of DTH reaction and was expressed as percent increment in thickness. The formula shown below was used to determine the increment [32] .
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