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Staining cells and Microscopy Lenzmeier Research Laboratory Protocol for staining cells with organelle-specific agents: 1. Preparing cultures for staining: 12-48 hours prior to doing your experiment you should split cells fresh onto containers that can be used for imaging using the confocal microscope—grow cells in 6 well plates or the small tissue culture petri dishes in 3 ml of complete medium per well/dish. Ideally your cells would be 50-75% confluent when carrying out the staining experiment. 2. Stains available: The stains below are available at BVU. Please note, you can stain with one red and one green fluorophore simultaneously in the same dish but should not use multiple red or multiple green fluorophores in the same dish. Structure Color Stain Mitochondria Red Mitotracker Deep Red FM Lysosomes Red Lysotracker Deep Red FM Cell Surface Green WGA-Alexafluor-488 Cell Surface Red WGA-Alexafluor-633 Nucleus (NAs) Green Syto9 Nucleus (NAs) Red NucRed Live-647 [Stock] 200 M 10 M 1 mg/ml 1 mg/ml 10 M N/A Solvent Aliquot amts DMSO 60 l DMSO 100 l PBS 125 l PBS 125 l DMSO 100 l PBS 2.5 ml bottles Location cold room cold room -20 freezer -20 freezer cold room cold room How to make stock solution 1 tube in 460 ul DMSO 50 l in 5 ml DMSO 5 mg bottle in 5 ml PBS 5 mg bottle in 5 ml PBS 10 l from tube in 5 ml DMSO Use tubes from manufacturer 3. Staining the cells: At the time you are going to stain the cells be sure to: Set out your PBS in the hood so it is at room temp when you do the washes immediately prior to microscopy. Turn on the confocal microscope and let it warm up for 30 minutes: o Follow the stepwise startup procedures for turning on the lasers and microscope. o Log into the computer (be sure to not let it do the drive scans) using the username “Administrator” with a password of “fluoview.” o Log into the imaging software use the username “Lenzmeier” with a password of “Lenzmeier”. Use the table on the right as a guide your staining and following the procedures on the next page. Color Stain Mitochondria Red Mitotracker Deep Red FM 6 l 30 min @ 37oC Lysosomes Red Lysotracker Deep Red FM 15 l 30 min @ 37oC Cell Surface Green WGA-Alexafluor-488 20 l 30 min @ 37oC Cell Surface Red WGA-Alexafluor-633 20 l 30 min @ 37oC 15 l 30 min @ 37oC Nucleus (NAs) Green Syto9 Nucleus (NAs) l per 3ml dish stain time/temp Structure Red NucRed Live-647 150 l (3 drops) 30 min @ 37oC Prepare a mix of stain/medium. For example, if you are staining 6 petri dishes with mitotracker deep red FM and WGA alexafluor 488: o Place 18 ml of medium (3 ml per dish being stained) into a sterile 50 ml tube o Add 36 l of mitotracker deep red FM (6 l per dish being stained; see table above) o Add 120 l of WGA-Alexafluor-488 (20 l per dish being stained; see table above) Mix the stain/medium tube well by inversion several times. Page 1 of 4 Staining cells and Microscopy Lenzmeier Research Laboratory Remove the original medium from the dishes being stained and add 3 ml of stain/medium into each dish. Incubate dishes 30 minutes at 37oC. Remove stain/medium and discard into waste Wash each dish gently with 3 ml of PBS and then remove PBS and discard into waste Add 3 ml of fresh PBS to each dish and proceed to the confocal microscope for imaging. 4. Obtaining images of the cells using the confocal microscope: A. Initial focusing with the transillumininator lamp: Turn on the transilluminator lamp using the button in the upper left corner of the Image Acquisition Control panel on the computer screen Push in the deflector on the microscope to the eye setting Set the filter wheel on the microscope to #6 (dict) Open the shutter by sliding the knob on the microscope from to O Place your sample on the microscope platform and use the 10X objective Focus using the coarse focus green buttons on the microscope until your cells are in view Use the fine focus adjustment knob until you feel your cells are focused appropriately. Turn off the transilluminator lamp by clicking the button in the upper left corner of the Image Acquisition Control Panel on the computer screen Pull out the deflector on the microscope to the camera setting Set the filter wheel on the microscope to #1 (LSM) Leave the shutter open on the setting open on the microscope (setting of O) B. Obtaining fluorescent images In the acquisition settings panel: Change the “Aspect Ratio” to either 512X512 or 320X320 Make sure the correct objective has been chosen (usually UPLAPO 10X NA:0.40) In the Image acquisition control panel: Make sure the box for “Kahlman” filter and “Sequential” are not checked in the bottom of this panel. Select the lamp from the dye list depending on your fluorophor by clicking on the green circle button (upper left corner third button down). You will need to click “all clear” and then scroll down in the dye list below, find your dye(s), double click the dye(s) you want, and then click the “Apply” button. The table below contains a list of what settings to use for each stain: Structure Color Stain Mitochondria Red Mitotracker Deep Red FM Lysosomes Red Lysotracker Deep Red FM Cell Surface Green WGA-Alexafluor-488 Cell Surface Red WGA-Alexafluor-633 Nucleus (NAs) Green Syto9 Nucleus (NAs) Red NucRed Live-647 Exitation l 644 nm 644 nm 495 nm 632 nm 485 nm 638 nm Emission l 665 nm 668 nm 519 nm 647 nm 498 nm 686 nm Microscope filter setting Alexafluor 647 dye Alexafluor 647 dye Alexafluor 488 dye Alexafluor 647 dye Alexafluor 488 dye Alexafluor 647 dye Page 2 of 4 Staining cells and Microscopy Lenzmeier Research Laboratory Once you’ve selected the dye(s) you want, close the dye list panel. Click on “XY repeat” to start viewing a live fluorescent image of your cells. You may not see anything…you will need to consider the following issues in obtaining your images: o You might need to focus using the fine focus on the microscope o You might need to alter the laser intensity by clicking changing the value for HV for that dye o You might need to alter the “gain” setting. Increasing this number makes the brightness (fluorescence) brighter but also increases the background noise. o You might need to alter the “offset” setting. Increasing this number makes the darks darker (decreases background but takes away from the signal. o You might need to alter all of the above settings to get the best photo. o You need to pay attention to bleaching of your fluorophors. Repeated excitation leads to a loss of fluorescence. Move quickly to focus to avoid photobleaching. Once you have an image you like, click the “repeat stop” button (the second button to the right of XY repeat. Record the settings you used in your lab notebook so this can be repeated at future times. In the acquisition settings panel: Set the “Aspect Ratio” to the desired resolution. Typically this will be 800X800 or 1024x1024. In the Image acquisition control panel: Click on the Kahlman filter box toward the bottom of the panel. You may wish to get an image with this to improve crispness, but it adds to bleaching because it takes longer to obtain the image. Try it and see if you want to use this for your images. Click on the “XY” button toward the top of the panel to obtain your image. Adjust settings accordingly for future images. Note: If wanting to simultaneously examine multiple fluorophors, optimize the settings for HV, gain and offset for each fluorophor and write them down. Then choose all the fluorophor excitation/emissions from the dye list and make sure the optimal HV, gain and offset settings correspond to each dye. When you do your image acquisition you will want to click on the “sequential” box at the bottom of the panel and then obtain your image using “XY.” C. Saving fluorescent images Go under “file” and save your image to your folder. Save it immediately as an .obi file. You will not be able to open this type of file on your computer but will want to save it so you can access all the quantitative information embedded in the file. To obtain images that you can open and process/quantitate on your computer, go under the file tab and “export as tif.” D. Transferring fluorescent images to your computer Use the usb flash drive to transfer the .tif files of your images from the hard drive to your computer. Page 3 of 4 Staining cells and Microscopy Lenzmeier Research Laboratory 5. Turning off the computer and microscope components: Close out of the software and transfer your files. Shut down the computer On the microscope: o Push in the deflector on the microscope to the eye setting o Set the filter wheel on the microscope to #6 (dict) o Close the shutter by sliding the knob on the microscope from O to o Place the microscope dust cover back over the microscope Follow the stepwise instructions next to the microscope for how to turn off the microscope components. Be sure to wait until the laser cooling fan shuts off before flipping its switch to off. Page 4 of 4