Hello, my Name is Rick Pasqua, student of the Stem Cell Engineering Lab here in the Department of Chemical and Life Sciences at the Virginia Commonwealth University. In this video, I will be demonstrating how our lab uses an inexpensive and self-made STO spin apparatus to create an observable monolayer of human pluripotent stem cells for the quantification of cell surface markers. Given that many IMMUNOCHEMICAL protocols are adapted for h PSCs grown in chamber slides as colonies problems can arise in characterizing the expression of the biomarkers at the individual cell level.
Having the cells in a monolayer aids not only in the visual analysis of these, but possibly also in quantifying their expression, the sinus spin apparatus our labs uses can be constructed using materials found in most labs only needs a small cell sample and doesn't need any type of specific reagent. And more importantly, it is able to reliably reproduce a spread of single cells for observation. Here are some of the things you'll need to construct the cytosine apparatus.
Plastic slides, since these don't necessarily have to be clean, you can Use old chamber slides if you wish. Pre cleaned glass slides. These need to be cleaned because this will be where the cell monolayer is created.
Filter paper, Any kind should Do, and micro pipette tips. One to 10 microliter tips are what our lab uses before construction can Begin. Holes should first be made into the plastic slides.
This can be done using a drill press. The diameter of the holes depends on the size of the micro pipette tip and should be about the width of the widest section of the tip used for a standard one to 10 micro pipette tip. This should be at around seven millimeters.
The amount of holes you create in the plastic slides completely depends on your intended use for it. For our lab's immunochemical staining, for instance, we usually use two holes, one for the positive stain and the other for the negative background control. These slides can be reused for an indefinite amount of times.
For the filter paper, simply cut out a piece of it to the parameters of the plastic slide. Use the holes of the slide as a reference for making holes on the filter paper. The size of the hole on the filter paper should be large enough so that the micro pipette tip can fit snugly as shown here.
We then need to cut out the appropriate number of micro pipette tips, one for each hole at approximately half its length. This will serve as the base of the glass slide for where the cellular mono layer will be created. Next, the filter paper will be put on top of this.
The filter paper will serve to absorb any of the excess medium from the suspension staining. This will then be topped off by the plastic slide that will serve as the apparatuses solid support. This can then be sealed off on all four sides with regular household tape.
Finally, whole micropipet tips are then inserted into the cut tips of the apparatus. This will be where the cellular samples following the suspension staining are placed into. It might also be helpful to initially mark where in the glass slide the cut tips are on for more convenient recognition.
The Immunochemical Suspension Staining protocol our lab uses involves first harvesting of stem cells into a single cell suspension, usually done by enzymatic ing, using trypsin or non enzymatic EDTA based cell dissociation buffers. The cells are then transferred on to one ml are two ml micro centrifuge tubes and their standard growth medium. These are then centrifuge at 200 Gs for four minutes.
The same centrifugation speed will be used for all consequent washing and spin down steps. The medium is then manually aspirated out, be extra careful while aspirating to ensure that the cell pellet is left undisturbed. This is then washed with one ml of PBS twice spinning down and aspirating out each time.
The next step involves fixing the cells using 4%pair formaldehyde or PFA reagents needed to make this or listed out in the accompanying writeup for this video. The cells are incubated in the 4%pair formaldehyde solution and left at room temperature for 10 minutes. It is recommended that this step be carried out prior to the ex extracellular suspension, staining for the use of the cyto spin apparatus.
Afterwards, the cells are washed twice with PBS to remove traces of the PFA. The cell should then be blocked to prevent unspecific staining. After the PBS washings at the previous step, aspirate out the wash and add one ml of the blocking solution.
This should be then left at room temperature for about 45 minutes. While waiting for the blocking solution, you can prepare the primary antibody by diluting it to the recommended dilution and blocking solution. Store this at four degrees Celsius.
After the cells have been in the blocking solution for about 45 minutes, spin down and aspirate this out and resus suspend them in the primary antibody solution. For the purposes of detecting nonspecific binding and background control, it is best to keep at least one sample as a negative. Were in no primary antibody applied and is simply resus suspended in PBS.
All the samples are left in room temperature for approximately one hour. If desired, fixed cells can be left in suspension in the primary antibody solution overnight and washed twice with blocking solution. The secondary antibody solution can be made while washing is done.
Secondary antibodies are usually light sensitive, so if possible dim lab lights and cover any containers used with aluminum foil, this is done to prevent fluorescence bleaching. Antifa reagents can be incorporated into the protocol, but care should be taken in their use as it can result in a higher background like the primary antibody diluted to the recommended concentration in blocking solution. Apply one vis to all samples whether or not primary was added to them after washing is completed.
Let this stand in a dark area such as inside a jar for about an hour. Wash the cells with one ml of blocking solution twice. The cell should now be ready for nuclear staining with DPI dye.
Prepare the DPI solution at a one to 10, 000 dilution in PBS initially, wash cells in one ml of PBS once, then resus, suspend in dpi, and let's stand in room temperature for about five to 10 minutes. Like the secondary antibody, DAPI is light sensitive, so make the necessary adjustments to prevent fluorescence, bleaching. Wash once with one ml of PBS and finally, reus.
Suspend again in one ml PBS. This should then be ready for use with a cyto spin apparatus. Place optim a maximum of 100 microliters of the single cell solution into the top of the micro pipet tips.
A lower amount is recommended though to prevent unnecessary overflow or dripping.Afterwards. Place a cyto spin apparatus and the counterbalance in a desk centrifuge and spin down at 1000 RPM for four minutes. Afterwards, carefully disassemble the cyto spin apparatus in a way that minimizes the chance of the dislodgement of the cells from the glass slide.
Verify that standing has taken place under fluorescence microscope before mounting. These are pictures taken of a monolayer of BG one V cell stained with SSCA four cell surface markers from the cyto spin apparatus. Notice how isolated the cells are from each other, making single cell observations and recording possible.
What's good about this protocol is that it is able to reproduce a monolayer cells that is feasible, cost effective, and easily adaptable for any routine analysis of stem cell cultures. So with that, I hope you draw this video and Find the protocol of use in your own research.
