Anti-CD105 antibody [MJ7/18] (PE) (ab93567)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
The first passages of ASCs were trypsinized (1% trypsin-EDTA, Sigma-Aldrich) and then centrifuged at 1,000 ×g for 5 min in the presence of 1% FBS to quench the enzyme. The cell pellets were resuspended in 1% FBS in PBS, filtered through a 70 μm cell strainer (BD Biosciences), and the cells were counted using a One Cell Counter (Wako Chemical).	10 μL anti-CD105 antibody [MJ7/18] (phycoerythrin; ab93567, Abcam) per 1,000,000 cells for 40 min. Antibody conjugated cells were subsequently filtered again through a 35 μm cell strainer (BD Biosciences). Cells were incubated with 5 μL 7-AAD staining solution (BD Biosciences) per 106 cells for 10 min and then sorted according to the expression of the cell surface markers

Upstream tips
The first passages of ASCs were trypsinized (1% trypsin-EDTA, Sigma-Aldrich) and then centrifuged at 1,000 ×g for 5 min in the presence of 1% FBS to quench the enzyme. The cell pellets were resuspended in 1% FBS in PBS, filtered through a 70 μm cell strainer (BD Biosciences), and the cells were counted using a One Cell Counter (Wako Chemical).
Protocol tips
10 μL anti-CD105 antibody [MJ7/18] (phycoerythrin; ab93567, Abcam) per 1,000,000 cells for 40 min. Antibody conjugated cells were subsequently filtered again through a 35 μm cell strainer (BD Biosciences). Cells were incubated with 5 μL 7-AAD staining solution (BD Biosciences) per 106 cells for 10 min and then sorted according to the expression of the cell surface markers

Publication protocol

The first passages of ASCs were trypsinized (1% trypsin-EDTA, Sigma-Aldrich) and then centrifuged at 1,000 ×g for 5 min in the presence of 1% FBS to quench the enzyme. The cell pellets were resuspended in 1% FBS in PBS, filtered through a 70 μm cell strainer (BD Biosciences), and the cells were counted using a One Cell Counter (Wako Chemical). Twenty microliters of purified rat anti-mouse CD16/CD32 (Mouse BD Fc Block, BD Biosciences) was added to 2 × 107 cells and the mixture was incubated at 4°C for 20 min. Cells were separated into 100 μL fractions at a concentration of 1 × 107 cells/mL and then incubated at 4°C with 0.5 μg anti-CD90/Thy1 [FITC.MRC OX-7] (FITC) per 1,000,000 cells (ab226; Abcam, UK) and/or 10 μL anti-CD105 antibody [MJ7/18] (phycoerythrin; ab93567, Abcam) per 1,000,000 cells for 40 min. Antibody conjugated cells were subsequently filtered again through a 35 μm cell strainer (BD Biosciences). Cells were incubated with 5 μL 7-AAD staining solution (BD Biosciences) per 106 cells for 10 min and then sorted according to the expression of the cell surface markers CD90 and/or CD105 using fluorescence-activated cell sorting (FACS) (FACS Aria II; BD Biosciences, Japan). Cells were collected in DMEM supplemented with 1% penicillin/streptomycin and 10% FBS. ASCs were sorted into four groups: CD90(−)/CD105(−), CD90(+)/CD105(−), CD90(−)/CD105(+), and CD90(+)/CD105(+); unsorted ASCs were used as a control.

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