This procedure uses live cell imaging to visualize the transfer of GFP labeled HIV across virological synapses between HIV expressing jerk CAT T cells and uninfected primary T cells. The cell to cell transfer assay starts with transecting jerk CAT T cells with HIV gag IG FP, an infectious fluorescent clone of HIV one, the transfected jerk cat cells expressing HIV gag. IG FP are then mixed with C-M-T-M-R labeled uninfected primary T cells and loaded into an imaging chamber where the transfer of GFP labeled HIV across the virological synapse can be observed.
The resulting data are analyzed using image analysis software to reveal the details of HIV transfer across T-cell virological synapses. Hi, my name's Benjamin Dale from the laboratory of Ben Chen in the Department of Medicine at the Mount Sinai School of Medicine. My name is Greg McNerney under laboratory of Dr.Thomas Huser at the NSF Center for Biophotonics at the uc Davis Medical Center.
And I'm Deanna Thompson, also from the lab of Dr.Thomas Huser. Today we'll show you a procedure for visualizing cell cell transfer of HIV one using live confocal microscopy. In this procedure, jerk hat cells are transfected with an infectious molecular clone of HIV only trained laboratory personnel working in certified biosafety.
Level two plus or biosafety level three tissue culture rooms can carry out the protocol shown here. Working with infectious HIV in imaging facilities must be approved by the local institutional biosafety office. Maintain the jerk cat cells to be used as donor cells at a concentration between two times 10 to the fifth and eight times 10 to the fifth cells per milliliter.
In jerk cat culture, media cell concentration in excess of eight times 10 to the fifth cells per milliliter results in a reduction in trans efficiency. To begin the procedure, transfect five times 10 to the six jerk cat cells with the viral plasmid, HIV gag IGFP Using the amax nucleo affection method as described in the accompanying written protocol, allow the cells to recover overnight. Peripheral blood mononuclear cells are purified from buffy coats of whole human blood by FICO density gradient centrifugation.
The primary CD four positive T cells are purified from the peripheral blood mononuclear cells by negative selection using a CD four positive T cell isolation kit. The negatively selected CD four positive T cells are collected from a magnetic column and cultured in our PMI with 10%FPS that is supplemented with 10 units per milliliter of recombinant human IL two one can also aliquot the isolated primary CD four positive T cells and freeze them with the aid of cryogenic freezing containers. The freezing containers are placed in a minus 70 degree Celsius freezer for a minimum of three to four hours.
The cells are then transferred to liquid nitrogen for long-term storage. Now that the jerk head cells are transfected and the target primary CD four positive T cells are isolated, proceed to cleanup and labeling of the cells on the day following the transfection. The jerk cells are now expressing the infectious fluorescent HIV construct.
When handling infectious samples, we wear a front closing lab coat and two pairs of gloves. Remove the cellular debris from the transfected cells by centrifugation over a fial high pack density medium. First dispense 1.5 milliliters of fial pack at the bottom of a 15 milliliter conical tube.
Then gently overlay the gradient material with the transfected jerk hat cells in a five milliliter volume of our PMI medium. At this point the transfected jerk hat cells are capable of producing infectious HIV. So all pipette tips that come into contact with the HIV expressing cells must be disinfected with 10%bleach solution.
Before discarding into a container within the biosafety cabinet, cap the tube and spray the outside of the tube with a solution of 70%ethanol and wipe off with a dry kim wipe centrifuge the cells at 400 Gs for 20 minutes at room temperature with the break off. After centrifugation, use a pipette to carefully remove the cells from the media fial interface and transfer the cells to a new 15 milliliter conical tube. Dilute the cells with our PMI medium to a total volume of 15 milliliters and centrifuge at 300 GS for 10 minutes.
At room temperature we suspend the pellet in three milliliters of jerk hat culture media and transfer to a two centimeter well of a six well plate. Place the plate in the tissue culture incubator until ready to use the following day. On the evening of the same day, label the primary CD four positive T cells used as target cells in order to distinguish them from the donor cells First pellet four times 10 of the six primary CD four positive T cells by spinning at 400 Gs for 10 minutes.
Then wash the cells with five milliliters of PBS and Resus. Suspend in two milliliters of PBS add cell tracker orange to a final concentration of 1.5 micromolar and incubate at 37 degrees Celsius for minutes. When the cells have finished incubating, add eight milliliters of complete chica media and centrifuge at 400 Gs for 10 minutes.
Re suspend the cells in three milliliters of complete media supplemented with 10 units per milliliter of IL two and place in the tissue culture incubator overnight. Following the overnight incubation, the donor and target cells are ready for the cell to cell transfer experiment. Before setting up the cell to cell transfer experiment, prepare the imaging chamber to buffer the platform against temperature fluctuations in the room.
And to block out background room light, a large black shroud is draped over the whole microscope heat setup, creating an insulated air pocket around the system. The imaging chamber is maintained at a stable 37 degrees Celsius using a thermostatic heater with a T type thermocouple tip placed next to the sample for temperature feedback. Preheat for 30 minutes prior to mounting the sample.
Spinning disc confocal imaging with an inverted optical microscope is used to create 3D confocal images of cell to cell transfer of HIV. All hardware and acquisition including a Z stage, are controlled by the and or IQ version 1.8 software scanning is performed by a CS U 10 spinning disc confocal unit that simultaneously uses more than 800 confocal spots to dramatically increase the scan rate to compliment its speed. The CS U 10 is paired with a highly sensitive electron multiplying charred coupled device camera to allow fast low light imaging, which reduces both photobleaching and phototoxicity.
The fluorescence light source is a multi wavelength argon krypton ion gas laser. An and or Oko optic tunable filter or A OTF is used to select the desired wavelengths, which are 488 nanometers and 568 nanometers here while controlling their power to further reduce photobleaching and to extend imaging time. The A OTF rapidly shutters the laser following each 15 to 30 millisecond exposure when the camera is not acquiring images, avoiding unnecessary exposure while the images are transferred from the E-M-C-C-D camera to the computer.
The laser light is incorporated into the spinning disc system using a 4 0 5 4 88 5 68 6 47 quad band Dichroic mirror fluorescent emission is separated by wavelength using a 560 nanometer long past diic mirror with an image splitter. The green channel is filtered using a 525 over 50 nanometer band pass filter and the red channel is filtered using a 609 over 54 nanometer band pass filter. The two channels are projected side by side onto the E-M-C-C-D camera.
While the imaging chamber is warming up, prepare the cells for the cell to cell transfer experiment. Use a hemo cytometer to count the HIV gag IGFP expressing ette cells. Place the hemo cytometer in a Petri dish to avoid direct exposure to the virus expressing cells.
Wash the cells with carbon dioxide, independent media and resuspend in live cell imaging media at a concentration of one to three times 10 of the seven cells per milliliter. The use of carbon dioxide independent media eliminates the need for a carbon dioxide environmental chamber. For imaging.
The cells also count, wash and resuspend the labeled primary CD four positive T cells as just shown for the jerk hat cells. Next, initiate cell to cell transfer mix 20 microliters of HIV gag IGFP transfected dur hat cells with 40 microliters of labeled primary CD four positive T cells and load 50 microliters into a tissue culture treated gas permeable. EBD micro chamber seal the chamber with plugs and secure the plugs by wrapping the plug EBIT D chamber interface with paraform.
Wipe down the imaging chamber with a chem wipe wedded with 70%ethanol and allow it to air dry briefly in the hood. Transport the sealed imaging chamber in a sealed container to avoid accidental exposure. Should the chamber be dropped in transit immediately after loading the IBD imaging chamber with cells, mount the device onto the microscope.
Take images of cell pairs using a 60 x 1.42 NA oil immersion objective As the high numerical aperture greatly improves resolution to maximize the speed of acquisition per 3D image stacks. Crop the recording region of the EM CD's camera down to the immediate area of a cell pair. Furthermore, a large Z step between 0.45 and 0.75 micrometers may also help speed up the acquisition under these conditions.
Images are continuously recorded at 20 to 60 minute intervals. Repeated imaging of the same sample spot can extend up to six hours with minimal photo bleaching when acquired. Each data segment is comprised of tens of thousands of images that may occupy a total of five to 20 gigabytes.
To facilitate the transport and analysis of the large files, each file is broken down and exported as one gigabyte TIFF image File segments. To analyze the acquired images, use any of several image analysis software programs. Consult the accompanying written protocol for details of image analysis, including intensity bleach correction and automatic and manual tracking of gag puncta.
Within 10 to 15 minutes of mixing, one can visualize HIV gag IGFP expressing jerk cat cells adhering to primary CD four positive T cells imaging. These conjugates one can assess the movements of gag puncta in infected cells as well as in target cells. After synapse, within 30 minutes to three hours, one may observe movement of gag IG FP puncta into target cells that are cell tracker labeled.
We've just shown you how to visualize cell to cell transfer of HIV between T-cells. When doing these procedures, it's very important to remember to observe all biosafety level two plus or biosafety level three precautions when handling samples that have been exposed to HIV expressing cells. Also remember to use well maintained JCA cells and endotoxin free DNA to optimize transfect.
So that's it. Thanks for watching and good luck with your experiments.
