Jan 10, 2022
spyDNA650 staining with CO2 independent media for live endothelium microscopy
This protocol is a draft, published without a DOI.
- 1MDC Berlin, AG Gerhardt
- AG Gerhardt
Protocol Citation: Emir Bora Akmeriç 2022. spyDNA650 staining with CO2 independent media for live endothelium microscopy. protocols.io https://protocols.io/view/spydna650-staining-with-co2-independent-media-for-b3m7qk9n
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: January 10, 2022
Last Modified: January 10, 2022
Protocol Integer ID: 56735
Abstract
"spyDNA650 staining with CO2 independent media for live endothelium microscopy"
Staining
Staining
Dilute 1:1000 SPY650 DNA in CO2 independent media(500 uL total volume for 2 slides, 220 uL per slide and incubate in incubator for 20 mins
Gently remove overnight media from the u-slides and add warmed dye-media mix. Check whether monolayer is preserved. incubate for 2-3 hours
Flow Unit Equilibration
Flow Unit Equilibration
In 7 mL Co2 independent media, dilute 3.5 uL SPY650 dye(1:2000 final concentration)
Add 3 mL media the leftwards syringe, 4 mL on the other
Set Ibidi Flow units up for quilibration inside microscope.
Start the heating of the microscope
Run for 1 hour, at 40 mbar and half the cycling time(about 5 secs), pay close attention to whether neither of the syringes run empty and do the clamp test.
Microscopy
Microscopy
Quickly bring one of the slides to the microscopy room without any washing step and plug it to the flow unit