Pacific blue mouse anti human cd3 clone ucht1

PBMCs were separated from whole blood using SepMate tubes (STEMCELL Technologies) with Histopaque (Sigma-Aldrich) gradient centrifugation. For each blood donor, 1,000,000 cells were stained at 4°C for 10 min with: Pacific Blue Mouse Anti-Human CD3 [clone UCHT1] (BD Biosciences, Franklin Lakes, NJ), Brilliant Violet 711™ anti-human CD56 (NCAM) Antibody [clone HCD56] (BioLegend, San Diego, CA), and PE Mouse Anti-Human HLA-C [clone DT9] (BD Biosciences). Sample data was collected utilizing a BD LSRFortessa flow cytometer and was analyzed with FlowJo v10.1. Unpaired t-tests of sample data were performed utilizing Prism 7.

BAL was performed as previously described [17 (link)]. Briefly, after light sedation and topical local anesthesia, a flexible fiber-optic bronchoscope (Olympus Optical Co., Tokyo, Japan) was passed trans-nasally and BAL was performed in a subsegmental bronchus in the middle-lobe. BAL was then performed by sequentially instilling and gently suctioning 50 ml of warmed (37°C) and sterile phosphate-buffered saline (PBS) solution five times. The BAL fluid aliquots were pooled and collected in a siliconized plastic vessel kept on ice at 4°C. The recovered volume as percentage of total instilled fluid is indicated in Table 1. The BALF was gently passed through a Dacron gauze (Milipore, Cork, Ireland) and centrifuged at 400 g for ten minutes at 4°C. The pellet was resuspended in PBS. The cells were counted in a Bürker chamber. The expression of cell surface antigens (CD3, CD4, CD8) and the CD4⁺ to CD8⁺ cell ratio were performed by flow cytometric analysis (BD FACSCanto II flow cytometer) using monoclonal antibodies against CD3⁺ (Pacific Blue Mouse Anti-human CD3, clone UCHT1; BD Biosciences), CD4⁺ (APC-H7 Anti-human CD4, clone SK3; BD Biosciences), CD8⁺ (AmCyan Anti-human CD8, cloneSK1; BD Biosciences) as previously described [18 (link)].