Impact of paclitaxel treatment on the Triple Negative Breast Cancer Cell line HCC1143

Data and code related to Zenodo repository: 10.5281/zenodo.11237850

Experimental goal:

Evaluate the impact of escalating paclitaxel dose on cell count, nuclear morphology and cellular outcome.

Methods:

Cells were plated at 3000 cells in 100ul of complete media per well in a 96 well plate (#08-772-225, FisherScientific). After 24 hours, an additional 100ul of either vehicle (0.1% DMSO) or paclitaxel containing complete media was added. After 72 hours cells were fixed with 4% Formaldehyde (#28908, ThermoFisher Scientific) for 15 minutes at room temperature, then permeabilized with 0.3% Triton X-100 (#X100-100ML, Sigma Aldrich) for 10 minutes at room temperature, then washed twice with PBS. Fixed cells were blocked with 1% BSA (A7906-100G, Millipore Sigma) in PBS for 1 hour at room temperature and then stained overnight with 1:100 anti-CDKN2A/p16INK4A+CDKN2B/p15INK4B-AF644 (#ab199756, Abcam), and 1:100 anti-cPARP-AF647 (#6987S, Cell Signaling Technology) or 1:500 anti-TUBB3-AF647 (#ab190575, Abcam) overnight at 4C. Each well was washed twice with room temp PBS then stained with 0.5ug/mL DAPI (4083S, Cell Signaling Technology) in PBS for 15 minutes at room temperature. Following DAPI staining, wells were washed once with PBS, then stained with 1:20,000 HCS CellMask in PBS (Orange: #H32713, Green: #H32714, Invitrogen) for 15 minutes at room temperature. Wells were washed twice with room temperature PBS and then 4 fields of view per well imaged on an InCell 6000 (GE Healthcare). Images were segmented with two custom Cellpose models to segment the nucleus (using parameters: diameter = 45, chan = DAPI, chan2 = Cellmask Orange) and cytoplasm (using parameters: diameter = 90, chan = Cellmask Orange, chan2 = DAPI). Image quantification was performed in R (v4.3.1) using EBImage (v4.42.0), and cells were annotated based on the number of distinct nuclei segmented within each cytoplasmic mask. 

Included files:

row_#_level_1.zip : 6 zip file containing original images from InCell 6000, one zip per row

level_2.csv : Data quantified to the nuclear level (cytoplasmic quantification is duplicates across multiplet nuclei)

level_3.csv: Data quantified at the cellular level including number of nuclei and stain intensities for segmented compartments

platemap.csv: Description of each well from the stained plate

cellpose_modelz.zip: Zip file containing the two CellPose models used for segmentation

image_quantification.rmd : R markdown file containing code for extracting and quantifying image intensities using the raw images (level_1) and segmentation masks created from cellpose.

本数据集与代码关联至Zenodo仓储：10.5281/zenodo.11237850

实验目标：
评估递增剂量的紫杉醇对细胞计数、细胞核形态及细胞结局的影响。

实验方法：
将3000个细胞以每孔100μL完全培养基的密度接种于96孔板（#08-772-225，FisherScientific）。培养24小时后，向每孔加入100μL含0.1%二甲基亚砜（DMSO）的载体培养基，或含紫杉醇的完全培养基。继续培养72小时后，使用4%甲醛（#28908，ThermoFisher Scientific）在室温下固定细胞15分钟，随后用0.3% Triton X-100（#X100-100ML，Sigma Aldrich）在室温下透化10分钟，再用磷酸盐缓冲液（PBS）洗涤两次。固定后的细胞用1%牛血清白蛋白（BSA，A7906-100G，Millipore Sigma）于室温下封闭1小时，随后在4℃下过夜孵育一抗：1:100稀释的抗-CDKN2A/p16INK4A+CDKN2B/p15INK4B-AF644（#ab199756，Abcam）与1:100稀释的抗-cPARP-AF647（#6987S，Cell Signaling Technology），或1:500稀释的抗-TUBB3-AF647（#ab190575，Abcam）。每孔用室温PBS洗涤两次后，使用0.5μg/mL DAPI（4083S，Cell Signaling Technology）于室温下染色15分钟。DAPI染色完成后，每孔用PBS洗涤一次，随后使用1:20000稀释的HCS CellMask染液（橙染：#H32713，绿染：#H32714，Invitrogen）于室温下染色15分钟。每孔用室温PBS洗涤两次后，使用InCell 6000成像系统（GE Healthcare）对每孔采集4个视野的图像。

使用两款自定义Cellpose模型对图像进行分割：细胞核分割参数为直径=45，通道=DAPI，通道2=Cellmask Orange；细胞质分割参数为直径=90，通道=Cellmask Orange，通道2=DAPI。图像定量分析采用R语言（v4.3.1）及EBImage包（v4.42.0）完成，并根据每个细胞质掩码内分割得到的独立细胞核数量对细胞进行注释。

包含文件：
row_#_level_1.zip：共6个压缩包，存储InCell 6000采集的原始图像，每板每行对应一个压缩包
level_2.csv：核水平定量数据集（细胞质定量结果在多细胞核样本中存在重复）
level_3.csv：细胞水平定量数据集，包含细胞核数量及各分割区域的染色强度信息
platemap.csv：染色实验所用96孔板的每孔信息说明文件
cellpose_modelz.zip：存储本次实验所用两款Cellpose模型的压缩包
image_quantification.rmd：R Markdown文件，包含基于原始图像（level_1）及Cellpose生成的分割掩码提取并定量图像强度的代码