293T OCMS and 293T cells were plated at 3 × 104 cells/well on German Glass Coverslips in 24-well plates in DMEM with 10% FBS. The following day, cells were transfected with 1 µg each recombinant 1G1 IgA heavy and light chain expression plasmids with X-tremeGENE 9 DNA transfection reagent (06 365 787 001, Sigma Aldrich). One day after transfection, the culture medium was replaced with 1 mL fresh medium with 1 μg/mL rabbit anti-human IgA mAb (ab193189; Abcam) and the cells were incubated for 24 h at 37 °C. The cells were washed 3 times with PBS 1% BSA, co-incubated for 1 h with 1:200 Alexa Fluor 647 conjugated goat F(ab’)2 anti-human IgA (2052-31; SouthernBiotech) and 1:200 Alexa Fluor 488 conjugated AffiniPure F(ab’)2 fragment goat anti-rabbit IgG (H+L) (111-546-144; Jackson ImmunoResearch) to detect the expression of IgA and OCMS respectively. The cells were then analyzed by confocal microscopy (see Section 2.4 ).
Ab193189
Manufactured by Abcam
Sourced in United States
Ab193189 is a laboratory equipment product manufactured by Abcam. It serves as a core function for research applications. The detailed description of this product is not available while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
2 protocols using ab193189
1
Visualizing IgA and OCMS Expression
2
Profiling Immune Cell Subsets by Flow Cytometry
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PBMCs were isolated using discontinuous Lymphoprep (Axis-ShieldPoCAs, Oslo, Norway) gradient centrifugation and were re-suspended in phosphate-buffered saline (PBS) containing 2% fetal bovine serum. Isolated PBMCs were collected by centrifugation, incubated with Fc-blocking antibody (purified anti-human CD16/32, BioLegend, San Diego, CA, USA) to prevent nonspecific binding and then incubated with the following antibodies (CD19: CF506236, BD Biosciences, CA, USA, 1:50; CD24: BDB563371, BD Biosciences, 1:50; CD27: BDB340424, BD Biosciences, 1:50; CD8: BDB564805, BD Biosciences, 1:50; IgA: ab193189, Abcam, CA, USA, 1:50) for 30 min on ice. After staining, cells were analyzed using CyAn-ADP (Beckman Coulter). Intracellular cytokine staining was performed by using the stimulation cocktail (eBioscience, San Diego, CA, USA) pre-stimulated for three hours as the manufacturer’s recommended protocol. Samples were analyzed by CyAn-ADP (Beckman Coulter) and data were processed using Flow Jo software (TreeStar, Ashland, OR).
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