Crystal violet staining & caliculation
Aim Stain live cells
Materials
0.2 um filtrated_0.1% crystal violet (0.1 g crystal violet dissolved into 2 ml EtOH + 98 ml ddw), filtration is to remove the aggregate not for sterilization.
PBS (not sterial OK)
Espon GT-X980 scanner (next to the odyssey and scanner also connect the same computer)
lightning LED viewer 5000A4 (in the right drawer under the Espon GT-X980 scanner)
Methods for 96-well dish
remove medium and wash once with 100 ul of PBS
add 50 ul of 0.2 um filtrated_0.1% crystal violet at R.T for 10 min
remove crystal violet and collect into special bottle because crystal violet is not good for environment
add 100 ul of PBS and then remove x3
remove completedly of the PBS (when PBS left in the well, edge stainning is blurred)
turn on the Espon GT-X980 scanner and software ESPONScan
put on the dish with opening the rid ( you don't need concern about contamination if you want use the rest of the well, it's OK)
then put the lightning LED viewer 5000A4 on it as a reflector board. (We don't need turn on the light switch.)
colour, 1200 dpi setting, use preview botton
check flip box and make ROI (region of interest)
push scan botton, confirm folder and file name and format. tiff
Caliculation
open Fiji ImageJ program
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Aoki, 20201113追記
こんな感じで実験
11/4 水
Lenti-Xを10 cm x 10枚(2.5x10^6 cells/dish)を用意 11/5 木
11/7 土
ウイルス液を回収、フィルター(0.42 um)、Untitled (20 mLウイルス液に6.66 mL)を加えて4度で60分放置、4度で1500 Gで45分遠心、上清を捨てて1 mL PBSでペレットを懸濁し(20倍濃縮)、-80度で100 uLずつ分注して保存。 11/9 月
朝、HeLaをcollagen coated 96 well plateに播種(1/20で)
11時にウイルス液を1x Polybreneこみで段階希釈して感染させた。
16時に培地交換
11/10火
11/12 木
を参考にした。
Crystal violet staining Total 40 mLを用意。
0.05% w/v Crystal Violet (0.01% Crystal Violetを20 mL)
1% ホルムアルデヒド(10% ホルマリン溶液を10.8 mL)
1x PBS(10x PBSを4 mL)
1% MetOH(400 uL)
DDW 4.8 mL(up to 40 mL)
培地を吸って、50 uL加える。20分室温放置。Crystal violet溶液をキムタオルに吸わせて捨てて、水道水で洗う。
乾燥させてスキャナーで画像取得(Odyssey PCを立ち上げ、スキャナーの電源を入れる。PCのスキャンのソフトを立ち上げて、プレビューで撮影範囲を決める。保存フォルダなどの設定をする)
結果
https://gyazo.com/446c3fd88d200d21e34b537f171b7714
ウイルス濃度が高いところは、毒性により細胞が死んでいる。
1/8が一番良さそう。
https://gyazo.com/4d17b31cd0ad706e778ebd8750c3dcce
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Remarks
Date :2019/11/18
Modified Date :
Author :Reina