Research
Centers and Institutes
External Designation Centers
Mississippi Center of Excellence in Perinatal Research
Research Resources Core B
Sub-Cores
Imaging Flow Cytometry and Flow Cytometric Analysis Core
Equipment

Equipment

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Amnis ImageStream MkII

The Amnis ImageStream^X MkII is a benchtop imaging flow cytometer system that combines the detailed cellular images and morphologic information of microscopy with the large sample size and phenotyping abilities of flow cytometry. The system has two CCD cameras, three lasers (Violet 405 nm, Blue 488 nm, Red 642 nm), and three imaging objectives (20x, 40x, 60x). The ImageStreamX MkII captures multiple images of each cell in flow, including brightfield, darkfield, and up to 10 fluorescent markers simultaneously. The image resolution depends on the magnification settings, with pixel sizes of 1 μm2, 0.25 μm2, and 0.1 μm2, respectively.

Data analysis is performed using the IDEAS software, which utilizes wizard-based and customized workflows to objectively quantify distinct cell populations, cellular features, and processes, including nuclear translocation, shape change, internalization, and apoptosis.

Applications include:

Intracellular co-localization
Nuclear translocation
Internalization and phagocytosis
Cell-cell interaction
Cell death and autophagy
Cell cycle and mitosis

For more information, visit the manufacturer's website: https://cytekbio.com/pages/imagestream

When planning an imaging flow cytometry experiment and choosing fluorochromes, it is important to know the fluorochromes' specific excitation and emission wavelengths, as well as the lasers, filters, and detectors available on the instrument. Below is the information for the Amnis ImageStream^X MkII.

	Amnis ImageStream^X MkII Configuration and Fluorochromes
Camera	Excitation Laser	Channel	BandpassFilter	Ideal dyes	Additional Fluorochromes
Camera 1	Bright field	Ch1		Bright Field	Bright Field
	Blue 488 nm	Ch2	528/65 (480-560)	FITC, AF488, GFP	CFSE, YFP, DyLight488, PKH67-membrane label, LysoTrackerGreen
		Ch3	577/35 560-595	PE	Cy3, DSRed, PKH67-membrane label SYTOX Orange
		Ch4	610/30 595-642	PE-TexasRed*	RFP, PE-AF610, eFluor625, Qdot625* Ch4 & Ch10 BF**
		Ch5	702/85 642-745	PE-Cy5*, PE-AF647	PI, 7AAD, PerCP-Cy5.5, eFluor 650, Draq 5
		Ch6	765/40 745-785	PE-Cy7*, PE-AF750	or use for side scatter (SSC)
Camera 2	Violet 405 nm	Ch7	457/45 435-505	DAPI, AF405, BV421	Hoechst, eFluor 450, DyLight 405, CFP, Live/Dead violet
		Ch8	537/65 505-570	BV510	AF430, eFluor506, Qdot 525, Cascade yellow
		Ch9	582/25 570-595	Bright Field	Qdot565, Qdot585, eFluor565
		Ch10	610/30 595-642		Qdot 605, Qdot625, eFluor625, BV605 Ch4 & Ch10 BF**
		Ch11	702/85 642-745		BV711, eFluor700 Qdot 705*
		Ch12	765/40 745-785		Qdot 800, BV786 or use for side scatter*
	Red 640 nm	Ch11	702/85 642-745	AF647, AF660, AF680	APC, Cy5, AF660, Draq 5, DyLight649, DyLight680, PE-AF647, PE-Cy5
	Red 640 nm	Ch12	765/40 745-785	APC-Cy7, APC-AF750	APC-H7, Cy7, APC-eFluor780, AF-750, DyLight750, PE-Cy7* PE- AF750* or use for side scatter

*It is important to remember that many fluorescent dyes are excited by more than one laser and fluoresce in multiple channels. It is very helpful to use a spectral viewer when planning an experiment.

** There are options to switch the Brightfield to different channels if needed (Ch1 & Ch9, Ch2 & Ch8, Ch 4 & Ch10, and Ch6 & Ch12).

Ideal fluorochromes for this instrument are AF488, PE, PE-TexasRed, SSC-Ch6, DAPI (or BV421), AF647, and APC-Cy7.