Hi, I am Hope Campbell of Flow Cytometry specialist from Blood Research Institute at Blood Center of Wisconsin. Hi, I'm STR Baso, a graduate student in Dr.Bonnie Dieter's lab. I'm also at Blood Research Institute of Blood Center of Wisconsin.
Today we're gonna be doing a procedure isolating cells using fluorescent activated cell sorting. We use this procedure in a laboratory to study the function of lymphocytes and myeloid cell populations. Let's get Started.
In this procedure B and CD four T cells will be purified from mouse cytes. The starting number of target cells should be calculated based on the number of cells needed and the recovery rate of the machine using this method. The typical recovery rate for B and CD four T cells is 85 to 90%The starting SP cyte population is approximately 60 to 70%B cells and 20 to 30%T cells.
Thus, if you start with 100 times 10 to the six cells, you'll be able to recover 50 to 60 million B cells and 20 to 30 million T cells before beginning facts. The solutions and reagents needed for the protocol should be placed on ice. These include staining buffer consisting of phosphate buffered saline with 3%fetal calf serum suspension buffer consisting of 25 millimolar HEPs prepared in Hank's balanced salt solution or HBSS containing 3%fetal calf serum A 25 millimolar hep B solution prepared in fetal calf serum staining antibodies and compensation beads.
This protocol also requires the following supplies and reagents. 15 milliliter conical tubes, 12 by 75 millimeter polystyrene or polypropylene flow tubes. A nylon cell strainer trien blue, and a hemo cytometer sorting collection tubes should also be prepared ahead of time to do this.
At 300 microliters of the he's FCS solution to flow tubes or one milliliter of the HEPs FFC S solution to 15 milliliter conical tubes. These should be placed on ice until the collection process begins. Begin by generating a single cell suspension of the desired cell population.
If the cells isolated, using this procedure will be cultured. The entire cell preparation should be performed in a tissue culture hood. Using sterile technique wash the cells once adding ice cold staining buffer and resus suspending the cells by inverting the tube several times.
Then centrifuge the cells for seven to eight minutes at about 250 GS four degrees Celsius. Washing conditions may vary with cell type and concentration. For cytes, wash up to 200 times 10 to the six cells with 10 milliliters of staining buffer.
Carefully remove the supernatant by aspiration so that the cell pellet remains intact. Then resuspend the cells in ice cold staining buffer at a concentration of up to 50 times 10 to the sixth per 100 microliters. Resuspend the cell pellet by flicking or gently vortexing the tube.
Then transfer 0.25 to one times 10 of the six cells to a new 12 by 75 millimeter polystyrene or polypropylene flow tube on ice. And set this aside to use later as the unstained control sample to the first tube, add primary antibodies to the cell suspension to achieve the desired final concentration. Here PE Texas's red conjugated anti B two 20 antibodies are used to stain B cells while ZI conjugated anti TCR R beta andor 700 conjugated anti CD four antibodies are used to stain T cells mixed by flicking the bottom of the tube or by light.
Vortexing then place the cells on ice and incubate in the dark for 20 to 30 minutes. Following the incubation, wash the labeled cells by Resus, suspending them in 10 milliliters of ice cold suspension, buffer and centrifusion as before, after careful removal of the supernatant. Repeat this step to wash the cells a second time.
Finally, resuspend the cells in ice cold suspension Buffer. If the primary antibodies are not directly conjugated to a fluoro four, add labeled secondary antibodies or strep AVID in conjugate to the cell suspension and again, incubate the cells on ice in the dark for 20 to 30 minutes following the incubation. Wash the cells twice with ice cold suspension buffer.
After resus suspending the cells in ice cold suspension buffer. Determine the cell concentration and viability using a vital dye such as trian blue and hemo cytometer. Adjust the cell concentration to 10 to 20 times 10 to the sixth cells per milliliter by adding suspension buffer to avoid clogging the fax machine with clusters of cells that may have died during the isolation.
Procedure and staining. Filter the cells through a drip strainer immediately before beginning the sorting protocol. The pore size of the strainer should be chosen depending on the cell type for cytes a strainer with a 40 micrometer pore size is used.
Collect the filtered cells in a 15 liter conical tube or 12 by 75 millimeter flow tubes depending on the volume. And place the cells on ice while the cytometer is set up for the sorting procedure. If using BD comp beads as single color positive controls, prepare one control tube for each color used in the assay plus an unstained negative control tube.
After thoroughly vortexing the bead suspensions add one, drop each of the negative and positive bead suspensions to a 100 microliter suspension buffer for each color used and the negative control. Next, add one microliter of the labeled antibody to each positive control tube. In this experiment, one tube is prepared using PE Texas'Red B two 20 antibodies.
One using fit ETCR beta antibodies and one using lor 700 CD four antibodies mixed by flicking the bottom of the tubes and incubate for 15 to 30 minutes in the dark at room temperature. Wash the beads once with two milliliters of staining, buffer and centrifuge at 200 Gs for 10 minutes. Remove the supernatant by aspiration and resuspend the bead pellet in 200 to 500 microliters of staining.
Buffer set up of the flow cytometer varies depending on the manufacturer and should only be performed by appropriately trained personnel. The cell sorting procedure here will be performed using the BD defects aria. Before sorting, select the appropriate nozzle depending on the cell type to be sorted.
The available sizes for the facts aria are 70 micrometers, 100 micrometers, and 130 micrometers. The nozzle size should be at least three times the size of the cell to be sorted for lymphocytes, the 70 micrometer nozzle is selected. Aseptic cell sorting can be performed using the instrument's aseptic sort option.
Using this option, the entire sheath path is decontaminated with 70%ethanol according to the manufacturer's instructions. Be sure to replace sheath filters and replace PBS with sterile PBS. Once the cytometer is ready to go, place the pre chilled collection tubes containing heaps FCS into the appropriate collection device.
Available collection devices can hold up to two 15 milliliter conical tubes. Four 12 by 75 millimeter tubes or four micro tubes. Use 12 by 75 millimeter tubes for collecting less than 1.5 times 10 to the sixth cells and 15 milliliter conical tubes for collecting 1.5 times 10 to the six to 4.5 times 10 to the sixth cells.
Place the appropriate collection device into the sort collection chamber. Direct the side streams into the installed collection device for multicolor sorts. The cytometer first be calibrated to account for spectral overlap between the detectors.
The example shown here illustrates the overlap between the emission spectrums of fit E and pe. This overlap shown in the dark shaded area is called spillover. Here PE spillover would be detected in the fit E channel and fit e spillover would be detected in the PE channel.
This spillover must be corrected for in order to eliminate the residual fluorescence of one floor four in another channel. The process of correcting for the spillover is called compensation to perform manual compensation. Use cytometry software to set up a template that includes a bi-variate plot to display forward scatter FSC and side scatter SSC and one histogram for each fluro that will be used.
The method to set up these plots varies depending on the software. Place the negative control com tube into the loading port of the cytometer and begin the acquisition of data as the sample is running. Adjust the FSC and SSC to place the population of interest on scale.
Then adjust the fluorescence PMT settings to place the negative population or autofluorescence in the far left hand portion of the histogram. Record the negative control tube. Remove the negative control tube from the cytometer loading port and replace it with the first positive control com tube.
Now begin acquisition. Repeat this process for each of the positive control tubes recording the data for each tube. For each positive control, draw an interval gate or box around the events recorded on the histogram.
Then draw another interval gate in the negative portion of the histogram. When doing manual compensation, take into account each fluro used in the experiment by adjusting the median of the positives until it is equal to the median of the negatives. To adjust the median, adjust the spectral overlap values, either higher or lower until the medians for each fluorescent parameter matches closely as possible.
Using the settings established with the positive and negative control samples, prepare for the sorting procedure by determining the gait settings for the experimental sample. Place the tube of cells to be sorted into the cytometer loading port and begin acquisition using a speed of nine to 10, 000 events per second. Select a dot plot to display forward scatter versus side scatter.
Forward scatter is a general indicator of cell size and is placed on the x axis while side scatter is an indicator of cell granularity and is placed on the Y axis. Now gate the population of interest. Once gates have been determined, check to ensure the temperature of the loading port of the cytometer is set to four degrees Celsius.
Then place the experimental sample tube into the loading port. Press the sort option and begin acquisition collection tubes should be placed on ice as they are filled. Continue sorting until the desired number of cells have been collected or until the sample tube is empty.
Then stop the sorting by selecting the sort button again and remove the sample from the loading port. Next, place the collection tube directly onto the loading port of the cytometer and acquire 500 to 1000 events. Using the same settings used for sorting for T cells, the sorting procedure increased the purity from 21 to 99%For B cells, the starting B cell population of 5%was increased to 98%after sorting.
So we have just shown you how to purify b and t-cell population from cytes by fax. Remember, facts can be used to purify any cell population if single cell suspension can be generated and appropriate antibodies are available. In addition, cells stack genetically with fluorescent markers can easily be purified without the use of antibodies.
Also, always remember to use the proper controls for your experiments and always check your flow cytometry laser configuration to make sure you're using the correct fluorochromes and make sure to use appropriate gates so that they're around your populations properly. So that's it. Thanks for watching and Good luck with Your experiments.
