Chander Raman, PhD J. Stewart New (Dr. Kearney s Lab) Using the ImageStream to Study the Immune System

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Darlene Annabel Simmons
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1 Please come to the Research in Progress Seminar Series WONDERFUL WORLD OF TECHNOLOGY event featuring Chander Raman, PhD J. Stewart New (Dr. Kearney s Lab) Using the ImageStream to Study the Immune System Thursday, November 6, 214, 12: 1: PM SHEL 515, 1825 University Blvd. Raw data welcome! You provide the science, and we ll provide the food.

The Amnis ImageStream is acquired via a successful Veterans Administration Shared Equipment Evaluation (ShEEP) Program

2 The Amnis ImageStream Imaing Flow Cytometer is ready for you to use in SHEL Rm 13A Please contact Shawn Williams or call to set up an appointment. The Amnis ImageStream is acquired via a successful Veterans Administration Shared Equipment Evaluation (ShEEP) Program application (Dr. Louis J. Dell Italia, MD, ACOS, Research and Development) It is operated by the Analytic Imaging and Immunoreagents Core of the Rheumatic Diseases Core Center (P3-AR48311).

3 USING THE IMAGE STREAM TO STUDY PHAGOCYTIC AND KILLING POTENTIAL IN MACROPHAGES J. Stewart New Laboratory of Dr. John Kearney UAB Dept. of Microbiology

4 Amnis ImageStream X Mark II Please visit the Amnis gestream.html

5 Optic Setup Lasers on the SHEL 13A ImageStream Mark II

Recommended dyes (based on optimal excitation and detection channels) are in boldface. *Many dyes will excite by more than one laser, and this can increase cross camera compensation.

6 Recommended dyes (based on optimal excitation and detection channels) are in boldface. *Many dyes will excite by more than one laser, and this can increase cross camera compensation. **Channel bandpass may change depending on which laser are on during an experiment. Values listed are assuming 45,488, and 642 laser excitation. 1 laser (488): ideal dyes are AF488, PE, PE-TxRed, PE- Cy5, PE-Cy7, SSC-Ch12, 2 laser (488,642): ideal dyes are AF488, PE, PE-TxRed, SSC-Ch6, and AF647, APC- Cy7 Available lasers in SHEL Rm 13A 3 laser (45,488,642): ideal dyes are AF488, PE, PE- TxRed, SSC-Ch6, and DAPI, AF647, APC-Cy7

7 IDEAS- Analysis software for the Amnis Image Stream

8 Analysis Wizards in IDEAS

9 Working with Features in IDEAS

10 Adjusting the Spot Mask

11 The Bright Detail Similarity Feature Select Mask Channels for colocalization

How to find and download the IDEAS ImageStream Analysis Software? 1. Please go to https://www.amnis.com/login. html 2. Please create a log-in account 3. Log-in 4.

12 How to find and download the IDEAS ImageStream Analysis Software? 1. Please go to html 2. Please create a log-in account 3. Log-in 4. The title page will be as shown 5. Please select the software downloads link 6. You are in the download page to download the software and many other tutorial and educational materials.

13 A Wonderful World of Technology Event: Using Image Stream to Evaluate Activation and Translocation of Signaling Molecules Chander Raman, Ph.D. November 6, 214

14 Acknowledgements Carlos Mier (UNAM) Yanna Ding Shawn Williams Funding: NIH (NIAID, NINDS), NIH grants to RDCC AR48311, AI27667

15 Does absence of CD5-CK2 signaling alter TCR induced activation in thymocytes? CD5 ΔCK2BD/ΔCK2BD CD5 WT/WT CD5 ΔCK2BD/ΔCK2BD extracellular domain + OTII TCR OVA transmembrane domain Pseudo- ITIM Y Y S S cytoplasmic domain CK2 Y Y Δ S X CK2 OTII.CD5ΔCK2BD/Δ CK2BD

16 Relative MFI perk Relative MFI perk Basal and OVAp induced active ERK is elevated in DP cells and CD4 SP thymocytes from CD5-CK2 signaling deficient mice DP cells p=.9 * OTII CD5WT + PBS OTII CD5 CK2BD + PBS OTII CD5WT + OVA OTII CD5 CK2BD + OVA DP Cells CD4+ cells * * OTII CD5WT + PBS OTII CD5 CK2BD + PBS OTII CD5WT + OVA OTII CD5 CK2BD + OVA CD4+ Cells

17 Flowcytometry provided a first step analysis to determine if there are differences in levels of active ERK. Questions: Is the active translocated to the nucleus and are there differences in cytoplasmic vs nuclear perk between WT and CD5-mutant? This can be quantitatively evaluated using ImageStream

18 Normalized Frequency Aspect Ratio_M1 Intensity_MC_CD8 ImageStream Analysis (PhosphoProtein Analysis) All Nucleus In Focus 1e5 Single cells 3 2 Nucleus in Focus Focused Nucleus Single cells 1e4 1e3-1e3 DN CD8+ DP 1.6-1e4 CD Gradient RMS_M7_Hoechst 1) Choose the Nucleus that are in Focus Area_M1 2) Choose the Single Cells by Using Area and Aspect Ratio -1e3 1e3 1e4 1e5 Intensity_MC_CD4PE 3) Gating of subpopulations)

19 perk: Imagestream Analysis OTII CD5WT DP Cells OTII CD5ΔCK2BD DP Cells OTII CD5WT CD4+ Cells OTII CD5ΔCK2BD CD4+ Cells

20 Normalized Frequency Normalized Frequency Normalized Frequency Normalized Frequency Normalized Frequency Normalized Frequency Normalized Frequency Using masks to define the cytoplasm and nucleus, we can determine the MFI of perk in nucleus, cytoplasm and whole cell. 1 DP 1 DP 1 DP MFI MFI MFI DP Nucleus e3 1e3 Intensity_MC_pERK 1e4-1e3 1e3 1e4 Intensity_Cytoplasm Only_pERK -1e3 1e3 1e4 Intensity_Nucleus Only_pERK 5 CD4+ 5 CD4+ 5 CD4+ CD MFI MFI MFI e3 1e3 1e4-1e3 1e3 1e4-1e3 1e3 1e4 perk perk in Cytoplasm perk in Nucleus MFI MFI MFI Ratio Nucleus:Total perk

21 M F I p E R K N u c le u s R a tio p E R K N u c le u s :W h o le C e ll M F I p E R K N u c le u s R a tio p E R K N u c le u s :W h o le C e ll Elevated perk in nucleus of CD5-CK2 signaling deficient CD4+ thymocytes 1 D P C e lls 1. D P C e lls 8 **** **** O T II C D 5 W T P B S O T II C D 5 C K II P B S 1 8 **** C D 4 + C e lls **** C D 4 + C e lls O T II C D 5 W T O V A O T II C D 5 C K II O V A O T II C D 5 W T P B S O T II C D 5 C K II P B S O T II C D 5 W T O V A O T II C D 5 C K II O V A. O T II C D 5 W T P B S O T II C D 5 C K II P B S O T II C D 5 W T O V A O T II C D 5 C K II O V A - Although perk levels are different, ratio of nuclear to whole cell is not different. - Ova immunization induces greater increase of perk in nucleus of WT DP thymocytes

Imaging Flow Cytometry (IFC)

Imaging Flow Cytometry (IFC) Dr. Yael Lupu-Haber Flow Cytometry Application specialist e-mail: yaelupu@technion.ac.il Tel: 04-8293676 LS&E site: https://isu.technion.ac.il/ Imaging Flow Cytometry - Outline