Last updated: 2022-10-06

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Aim

The aim of this test is to assess Matrigel recovery with increased wash and transfer ## Researcher ML ## Previous test

Readout

Hoechst, ImageIt and Bright Field of full well at 4X on Cytation 10 Primary readout is about Spheroid counts Secondary readout is a deeper dive into whether spheroid size or Matrigel percentage made a difference in recovery

Required R packages

data.table, DT, platetools, reshape2, tidyverse, patchwork

# load the required packages
library(data.table)
library(DT)
library(platetools)
library(reshape2)
library(readxl)
library(tidyverse)
library(patchwork)
library(here)
library(htmltools)


# check if here() correctly identified the directory
here::here()

[1] "/home/mli/ML_Mac-Seq/Matrigel_recovery_dev/Matrigel_recovery_test_220913"

# set the file prefix

prefix <- "ML_Mac-Seq_Matrigel_recovery"

Screen details

Screen date (yyyy-mm-dd)

2022-09-13

Plate overview Z proj 3 channels prior to extraction

Plate overview Z proj 3 channels post to extraction

Known technical issues

Previous test yielded around 75% recovery rate.

Data cleaning

Formatting and Preprocessing

The raw data was read into R Studio.

Data processing

Matrigel Recovery

Percentage Recovered Datatable

Comments: By counting the spheroid objects prior and after extraction procedure in each well, I will be able to estimate the recovery rate.

The nice thing about our recovery method is that spheroids largely stay intact.

The challenging is during the image processing step, 1)some wells have too many spheroids and others have big clumps of them, 2) each spheroid no longer have the nice smooth “look”, and they sit on top of each other. They make the object segmentation very challenging. 3) Post recovery some spheroids did fall apart inflating the object counts 4) Even with PBS added back to the well, huge bubbles still occur.

Percentage Recovered by count

Comments: Recovery yield calculated by object count should be taken with a pinch of salt. The reason being small spheroid + super confluent wells are challenging to segement properly (source plate prior to recover) Cells falling apart due to mechanical disturbance will be now counted as objects, inflating the object numbers (source plate post to recover ) So the object count in the Prior is designed to be an underestimate, and Post is designed to be an overestimate.

Percentage Recovered by Area Occupied Ratio

Comments:

One way to get around is to use the area occupied measurement. The idea is to measure the area occupied in the prior and post recovery wells, use the prior to normalise the delta in area occupied.

Check if big spheroids are not recovered