Fitc mouse anti human hla abc

H9‐derived MSCs and human bone marrow‐derived MSCs (Lonza, Walkersville, MD,
http://www.lonza.com) were grown to confluence, harvested by using 0.25% trypsin/EDTA, and resuspended in buffer containing phosphate‐buffered saline (PBS), 2% HEPES buffer, 2% FBS, and 0.1% bovine serum albumin (BSA) as previously described 43. Cells (1 × 10⁶) were incubated with phycoerythrin (PE) mouse anti‐human CD90, PE mouse anti‐human CD73, fluorescein isothiocyanate (FITC) mouse anti‐human CD44, FITC mouse anti‐human CD45, FITC mouse anti‐human HLA‐ABC, PE mouse anti‐human CD29, PE mouse anti‐human CD166, PE mouse anti‐human HLA‐DR, FITC mouse anti‐human CD105, or FITC mouse anti‐human CD31 (BD Biosciences, San Jose, CA,
http://www.bdbiosciences.com). Nonspecific fluorescence was determined by using isotype‐matched monoclonal antibodies. A total of 10,000 events were collected on a BD fluorescence‐activated cell sorting Calibur Flow Cytometer instrument by using CellQuest software (BD Biosciences). Analyses of results and corresponding graphs were generated by using FlowJo software (Tree Star, Ashland, OR,
http://www.flowjo.com) 434445.

TC-1 cells were stained with fluorescein isothiocyanate (FITC) conjugated anti-mouse H-2Kb (clone AF6–88.5; BD) and mouse (BALB/c) FITC-IgG2a κ (BD) was used as an isotype control. Human cell lines were stained with FITC mouse anti-human HLA-ABC (clone G46–2.6; BD) and a FITC mouse IgG1κ antibody (BD) was used as an isotype control. Data was collected using an Epics XL-MCL flow cytometer (Beckman Coulter, Inc., Brea, CA). Data was analyzed using FlowJo Software (Tree Star, Inc., Ashland, OR). Results represent the change in mean fluorescent intensity (Δ-MFI) ± standard deviation corrected for mean expression of isotype controls and normalized to expression of non-CICs. Positive expression ±standard deviation was similarly corrected for expression of isotype controls. Expression of MHC-I was also assessed following 24 h of exposure to 500 units/mL recombinant mouse IFN-γ (R&D Systems, Minneapolis, MN).