Antibody Hybridization Dataset Using Human Leukocyte Antigen (HLA) Probes and Epithelial Cells (Buccal and Contact Epidermal)

Biological samples were collected from volunteers using the following protocol approved by the VCU-IRB (#HM20000454_CR). For contact epithelial samples contributors rubbed a sterile polypropylene conical tube (P/N 229421; Celltreat Scientific) for five minutes using their entire hand (i.e., palm and fingers). Cells were then collected from the surface with six sterile pre-wetted swabs (P/N 22037924; Fisher Scientific) followed by two dry swabs. To elute the cells into solution, the swabs were manually stirred then vortexed for 15 seconds in 10 mL of ultrapure water (18.2 MΩ∙cm). Buccal cells were collected by swabbing the inner cheek surface with a cotton-tipped swab. As before, swabs were mixed in 10mL of ultrapure water to create a cell solution used for subsequent hybridization experiments. For antibody hybridizations, three milliliters of each cell solution were centrifuged at 5,000xg for five minutes. The supernatant was decanted and the pellet was resuspended in 100 µl PBS buffer and 1 µL of Human Fc Receptor block (Cat# 130-059-901, Miltenyi Biotec) to increase the specificity of antibody binding before reaction with HLA probes. This was allowed to incubate at room temperature for 10 minutes. Cells were incubated with mouse anti-human monoclonal antibody (mAb) HLA-ABC-FITC (Cat# 311403, BioLegend) or HLA-A02 (Cat#343303, Biolegend) for 30 minutes. Cells incubated with anti-mouse IgG2a-FITC (Cat# 343303, BioLegend) for 30 minutes served as the isotype control for these experiments. Cells were then washed once in 1x FACS buffer [PBS supplemented with 2% Fetal Bovine Serum (FBS, Cat# 100-106, Gemini BioProducts) and 10% Sodium Azide (Cat# S2002, Sigma-Aldrich)] and re-suspended in the same solution until flow cytometry analysis.Flow cytometry analysis was performed on the BD FACSCanto™ II Analyzer (Becton Dickinson) equipped with 488nm and 633nm lasers. Channel voltages were set as follows: Forward Scatter (FSC, 150V), Side Scatter (SSC, 200V), Alexa Fluor 488 (FITC, 335V), Phycoerythrin (PE, 233V; PE-Cy5, 300V; PE-Cy7, 400V), and Allophycocyanin (APC, 250V). Certain samples were analyzed using the BD FACSAria Ilu (Becton Dickinson) using the same voltage settings.<br>Flow cytometry source data for all samples are provided in Flow Cytometry Standard (.fcs) format files. Data files are labeled by date of collection, Sample ID, cell type, and the antibody probe used. Isotype and unstained controls are included.

本数据集的生物样本均按照弗吉尼亚联邦大学伦理审查委员会（VCU-IRB, #HM20000454_CR）批准的实验方案，从志愿者处采集。
针对接触上皮样本，受试者用整只手（手掌与手指）摩擦无菌聚丙烯锥形管（货号229421；Celltreat Scientific）共计5分钟。随后使用6支预湿无菌拭子（货号22037924；Fisher Scientific）从管表面采集细胞，再辅以2支干拭子完成采集。为将细胞洗脱至溶液中，手动搅拌拭子后，将其置于10 mL超纯水（电阻率18.2 MΩ∙cm）中涡旋振荡15秒。
颊细胞（buccal cells）的采集采用棉拭子擦拭脸颊内侧表面的方式。同前述操作，将拭子置于10 mL超纯水中混匀，制备得到细胞悬液用于后续杂交实验。
针对抗体杂交实验，取每份3 mL的细胞悬液，以5000×g离心5分钟。弃去上清液后，将沉淀重悬于100 μL磷酸盐缓冲液（PBS）中，并加入1 μL人Fc受体阻断剂（货号130-059-901，Miltenyi Biotec）以提升抗体结合特异性，随后与HLA探针进行反应。该体系于室温下孵育10分钟。将细胞与小鼠抗人单克隆抗体（monoclonal antibody, mAb）HLA-ABC-FITC（货号311403，BioLegend）或HLA-A02（货号343303，BioLegend）孵育30分钟。以仅与抗小鼠IgG2a-FITC（货号343303，BioLegend）孵育30分钟的细胞作为本实验的同型对照。
随后用1×流式细胞术缓冲液[PBS添加2%胎牛血清（Fetal Bovine Serum, FBS, 货号100-106，Gemini BioProducts）及10%叠氮钠（货号S2002，Sigma-Aldrich）]洗涤细胞一次，并重悬于相同缓冲液中，直至开展流式细胞术分析。
流式细胞术分析使用搭载488nm与633nm激光器的BD FACSCanto™ II流式分析仪（Becton Dickinson）完成。通道电压设置如下：前向散射光（FSC, 150V）、侧向散射光（SSC, 200V）、Alexa Fluor 488（FITC, 335V）、藻红蛋白（PE, 233V；PE-Cy5, 300V；PE-Cy7, 400V）及别藻蓝蛋白（APC, 250V）。部分样本采用BD FACSAria Ilu（Becton Dickinson）完成分析，使用相同的电压设置参数。
所有样本的流式细胞术原始数据均以流式细胞术标准格式（Flow Cytometry Standard, .fcs）文件提供。数据文件以采集日期、样本ID、细胞类型及所用抗体探针命名，包含同型对照及未染色对照样本。