This video demonstrates a procedure for stimulating and staining peripheral blood mononuclear cells or pbmc. For multicolor flow cytometry analysis, Alec Watts of pbmc isolated from whole blood are placed in tissue culture plates. Cells are chemically or biologically stimulated to produce an immune response.
Following stimulation, a cocktail of antibodies is added to identify cell types based on expressed surface markers. The cells are then permeated and a different cocktail of antibodies is added to detect the expression of intracellular cytokines, cells are then analyzed by facts to determine the cytokine profiles for specific subsets of pbmc. Hi, I'm Amy Courtney from Dr.Jagan SAS's lab in the Department of Immunology at the University of Texas MB Anderson Cancer Center.
Hi, I'm Hong her also from the lab. Today we'll show you a multi cytometry analysis of T-cell responses. We use this procedure in our lab to study cellular immune responses in Reus Maca.
So let's get started. Begin this protocol using a hemo cytometer to determine the viable cell counts of pbmc from Reuss Maca by the Trian Blue Dye exclusion method. Working in a laminar flow hood reus.
Suspend the cells in complete medium at a concentration of 10 million cells per milliliter for non-specific stimulation at 100 microliters of the cell suspension into each well of a 96 well tissue culture plate. Then add 100 microliters of medium containing PMA and CIN to bring the total volume to 0.2 milliliters with a final concentration of 50 nanograms per milliliter PMA and 500 nanograms per milliliter CIN for negative control wells, add 100 microliters of complete medium alone to each. Well incubate the cells at 37 degrees Celsius in the humidified 5%carbon dioxide atmosphere after 1.5 hours.
A felden A to the culture at a final concentration of 10 micrograms per milliliter and incubate for an additional 4.5 hours. The felden A is added to trapp cytokines in the gold G apparatus to increase visibility with immuno staining, following stimulation centrifuge to remove medium and then wash by adding 200 microliters of cold flow wash, buffer and centrifuge at low speed. Repeat this wash, then resend in 50 microliters of flow wash buffer.
The cells are now ready to be stained for expression of surface markers to each well of the resuspended cells. Add one microliter of live dead fixable aqua fluorescent reactive dye prepared according to the manufacturers and S instructions. Incubate at four degrees Celsius in the dark for 30 minutes while the cells are incubating.
Prepare the staining solutions that will be used for facts in micro tubes. First, prepare a standing cocktail to include the following appropriately. Titrated fluorescence labeled monoclonal antibodies, anti CD three PE SCI seven, anti CD eight Alexa 700 anti CD 28 per CCP SCI 5.5, anti CD 95 A PC and anti CD four Pacific Blue Inflow Wash Buffer.
Next, prepare compensation controls, which will be used to determine the spillover coefficients of each individual stain into other detectors by preparing a tube of each fluorescently conjugated antibody individually as a single color stain. Finally, prepare fluorescence minus one or FMO stains by combining all reagents but the one of interest. This will be used to determine the boundary between a positive and negative population by duplicating autofluorescence levels and data present in fully stained samples.
After the cells have incubated in the live dead stain, wash them once with cold flow wash buffer to remove excess dye, aspirate the wash buffer. Then add the prepared staining solutions. Incubate the cells at four degrees Celsius in the dark for 30 minutes.
Wash the stained cells with cold flow wash buffer to remove excess antibody. Then add 100 microliters of BD fixation permeation solution for at least 10 minutes. Store the cells at four degrees Celsius in the dark up to 24 hours until they are used or continue on to label the intracellular cytokines to label intracellular cytokines.
First perme the cells in 100 microliters of one X perm. Wash buffer incubate for 10 minutes at four degrees Celsius in the dark while the cells are incubating. Prepare the staining antibodies, compensation controls and FMO controls in one x perm wash buffer as before.
After the cells of incubated for 10 minutes, spin down and remove the one x perm wash buffer. Add the appropriate staining antibodies. Then incubate the cells and antibodies for 60 minutes at four degrees Celsius in the dark following the incubation.
Wash the cells two times with the permeation solution. After the final wash, resus suspend the cells in 1%Paraform aldehyde in DPBS to fix the cells. Store the cells at four degrees Celsius in the dark until they are used for up to 24 hours.
Once cells have been stained, use an LSR two flow cytometer or cyan a DP flow cytometer to perform facts. Then analyze the data using FlowJo or similar software. PBMC composition in reuss Maca was determined using fax analysis.
This figure shows the gating scheme utilized for the analysis of the different T-cell subsets from a representative animal. The lymphocytes were first gated via forward scatter FSC versus side scatter SSC and then live lymphocytes were identified based on SSC and AQUA negative population. The T cells were then identified by CD three expression CD four positive, CD eight negative, and CD four negative CD eight positive populations within the CD three positive T cell population were also determined.
The CD four positive and CD eight positive T cells were further distinguished into different subsets on the basis of CD 28 and CD 95 expression as naive central memory and effector memory cells. This figure shows interferon gamma and IL two production in Unstimulated and pma. CIN stimulated CD four positive T cells and subsets.
Note the distinct cytokine profiles elicited by stimulation. In each subset. We have to show you how to perform multi flow cytometry analysis of pbmc.
When doing this procedure, it's important to remember to set up both single color compensation controls and fluorescent minus one controls. Both of these controls are necessary for proper analysis of flow cytometry data. Please refer to our written protocol for more information on choosing compensation controls.
Also, please remember that when using fluorescent labeled antibody, it's important to cover the samples to protect from light exposure. So that's it. Thank you for watching and good luck with your experiments.
