project hollywood

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Project Hollywood Fred Martin BIOSC. 3 Dr. Giorgi 4/30/2010 Specimen used: Buccal cells stained with ER Tracker Imaging systems used: Leica SP5 Objectives used: Leica SP5 HCX PL APO CS 40.0x/0.92 IMM- OIL Note: metadata detail presented on last page

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Live Cell imaging project using vital dye ( ER Tracker Green - Invitrogen)

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  • 1. Project Hollywood Fred Martin BIOSC. 3Dr. Giorgi 4/30/2010 Specimen used: Buccal cells stained with ER TrackerImaging systems used :Leica SP5 Objectivesused:Leica SP5 HCX PL APO CS 40.0x/0.92 IMM-OILNote: metadata detail presented on last page

2. Specimen Info: Buccal cells: Human cheek cells are squamosal epithelial cells which form the inner cheek wall lining. Morphology: Thin, flat polygonal cells which form a surface lining in the cheeks interior with irregular boundaries. Cell are arranged edge to edge. Unstained, the cells have a prominent nucleus, and little else visible at 100-400x. Stained with methylene blue, the plasma membrane borders become more apparent, as does the nucleus and cytoplasm. Smaller internal organelles may be apparent, but difficult to know what it is, or if its just fragments of your last meal. Typical morphology of simple stained of human buccal cells Image source:homebiology.blogspot.com/2009/05/cells.html Cell membrane Cytoplasm Nucleus 3. INFO ON LIVE LABEL:ER-Tracker Green (BODIPY FL glibenclamide) *for live-cell imaging* Cat. No. E34251TheER-Tracker Greendye is cell-permeant, live-cell stain that is highly selective for the endoplasmic reticulum (ER) that when stained using the protocol provided, the staining pattern is partially retained after fixation with formaldehyde. This stain consists of the green-fluorescent BODIPY FL dye and glibenclamide. Glibenclamide (glyburide) binds to the sulphonylurea receptors of ATP-sensitive K+ channels which are prominent on ER; the pharmacological activity of glibenclamide could potentially affect ER function. Variable expression of sulphonylurea receptors in some specialized cell types may result in non-ER labeling. 1.3 View/image cells: Required filter sets DyeEx/Em *Filter ER-Tracker Green dye504/511FITC BODIPY FL Cell Preparation and Staining 1.1 Prepare staining solution.Dilute the 1 mM stock solution to the final working concentration. We recommend working concentrations of 100 nM1 M for ER-Tracker Blue-White DPX and ~1 M for ER-Tracker Green and ER-Tracker Red dyes. To minimize potential labeling artifacts, use the lowest dye concentrations possible. Best results are obtained when staining is performed in Hanks Balanced Salt Solution with calcium and magnesium (HBSS/Ca/Mg, Gibco cat. #14025-092) at 37C/5% CO2. 1.2 Stain the cells . For adherent cells, remove the medium from the culture dish, rinse with HBSS, and add prewarmed staining solution. Incubate the cells for approximately 1530 minutes at 37C. Replace the staining solution with fresh probe-free medium and view the cells using a fluorescence microscope. If the stained cells are to be fixed, refer to the fixation steps below for the appropriate dye. MFG RECOMMENDED LIVE LABEL PROTOCOL: Source: Invitrogen website 4. Live Cell Imaging Working Protocol Fred Martin4/23/10 Specimen Cells:(1) Hela cells from Molecular Foundry(2) Alternative: Buccal cells Dye: ER-Tracker Green (BODIPY FL glibenclamide) Molecular Probes/Invitrogen #E34251 for live-cell imaging;highly selective for the endoplasmic reticulum 100u glot 497266 Dye Preparation:Note:Dye is supplied as 100 ug of lyophilized material.Step1:Prepare a 1 mM stock solution by dissolving the contents of the vial in 128 uL of anhydrous DMSO; Note: had to make high conc & dilute due to only ~30uL of DMSO available Tobeys dye prep. Step2:Separated into 2 working aliquots,and the unused portion of the ~4 mM solution to be stored frozen with desiccant. Stored conc stock in freezer(no dessicant) in 1.5mL label E-tube Cell Preparation and Staining Step3: Dilute staining solution to working conc.Dilute the ~4 mM stock solution to the final recommended working concentration of ~1u MUse Hanks Balanced Salt Solution with calcium and magnesium at 37C/5% CO2. (HBSS best results/low artifacts) Step4: Stain the cells.For HeLa cells, aspirate the medium from the culture dish;Rinse with HBSS for 1 min Add prewarmed staining solution.Incubate the cells(dry incubator, no CO2) for approximately 25 minutes at 37C.(actual was range from 35-42deg) Replace the staining solution with fresh probe-free, non-phenol red medium (used HBSS) Step5: View/image cells: Required filter sets DyeEx/Em *Filter ER-Tracker Green dye504/511FITC BODIPY FL 5. Representative Imagefrom Z-stack I chose this image because it shows both cells that took the stain, and those that did not; and for those that did, you can see what I perceive to be SER and RER distribution, also ER peri-nuclear membrane staining and interesting channels across that membrane 6. Complilation of Imagesfrom Z-stack Scale factor = 0.25 Start slice = 5 End slice = 68 Increment = 4 7. Buccal Cells: Series001 , AVIw/ ER Tracker From 77 sliceZ-stack 8.

  • Comments on process:
  • Lack of materials required some improvisation, but still good result
  • Decided to keep it simple and try 1 channel staining only and go for quality
  • Was not as difficult as I had imagined/perceived
  • Touching slide to coverslip (sitting on top of chamber) was easier than fishing coverslip out of chamber
  • Incubation control was poor due to poor incubator & lack of coordinated use
  • Observation of specimen:
  • I believe most cells were likely not viable by the time imaging commenced, but somewere during the staining stage as clear differences in uptake of stain are seen; took most of the live Hollywood out of the project, but good data no less
  • There do appear to be differential patterns the ER staining (proximal vs distal to nucleus) Speculate this may represent SER vs RER ? Differences in amount/location of binding sites?
  • The closeup zoom of the nucleus shows peri-nuclear staining and resolves some interesting features on the nuclear membrane surface; consistent with idea that parts of the ER is contiguous with the nuc. Membrane
  • See extra images that follow

9. Buccal Cells ER Tracker, Max Projection 33 slice Z-stck Note: peri-nuclear staining differences Dead cells? RER? SER? 10. Buccal Cells Series013 ER Tracker, Max Projection 54 slice Z-stck, 3.7 zoom factor Note: peri-nuclear staining differences Peri-nuclear membrane staining with apparentchannels / invaginations showing 11.

  • Pinhole [m]95.5 m
  • Pinhole [airy]1.00
  • Size-Width 246.0 m Size-Height 246.0 m
  • Size-Depth9.0m
  • StepSize0.12 m
  • Voxel-Width 481.5 nm Voxel-Height 481.5 nm
  • Zoom1.0Scan-Direction1SequentialMode0Frame-Accumulation 1
  • Frame-Average1 Line-Average1Resolution8 bits Channels = 1
  • Format-Width 512 pixels Format-Height512 pixelsLine-Accumulation1
  • Number of Sections 77
  • Hardware Details
  • PMT 1ActivePMT 1 (Offs.)-1.2 % PMT 1 (HV)646.7PMT 1 (HV_Unit)V
  • PMT2 Inactive PMT 3Inactive PMT Trans Inactive
  • Excitation Beam Splitter FWTD 488/543/633
  • Laser (Argon, visible)OnLaser (Argon, visible) (Power) 20 % Laser (HeNe 543, visible)On Laser (HeNe 633, visible)On
  • Scan Field Rotation0 degrees Scan Speed 400 Hz
  • Objective HCX PL APO CS 63.0x1.40 OIL UV Numerical aperture (Obj.)1.40Refraction index1.52

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