CellRapeutics™ Fixation/Permeabilization(CART-023CL)


Product Name
CellRapeutics™ Fixation/Permeabilization
Cat. No.
CART-023CL
General Description
For Intracellular Cytokine Analysis.
Simplify the preparation of cells for intracellular staining of cytokines.
Enable one step fixation and permeabilization of cells.
Components
Fixation/Permeabilization solution (125 ml) and Perm/Wash Buffer (100 ml)

This kit enables the fixation and permeabilization of cells which is necessary for staining intracellular cytokines with fluorochrome-conjugated anti-cytokine antibodies. The kit provides two reagents, fixation/permeabilization solution and Perm/Wash Buffer. After cell fixation and permeabilization, the Perm/Wash Buffer is used to wash the cells and to dilute the anti-cytokine antibodies for staining.
Procedure
A. Stimulation of Cells Various in vitro methods have been reported for stimulating cytokine producing cells. Polyclonal activators have been particularly useful for inducing and characterizing cytokine-producing cells. These include the following: phorbol esters plus calcium ionophore or ionomycin, phytohaemagglutinin, Staphylococcus enterotoxin B, and monoclonal antibodies directed against subunits of the TCR/CD3 complex (with or without antibodies directed against costimulatory receptors such as CD28).
Note: It has been reported that cell activation with PMA alone causes a transient loss of CD4 expression from the surface of mouse T cells. Cell activation with PMA and calcium ionophore together has been reported to cause a greater and more sustained decrease in CD4 expression, and also a decrease in CD8 expression in mouse thymocytes and mouse and human peripheral T lymphocytes. 1. Procedure for Using GolgiStop Protein Transport Inhibitor (contains monensin)
Add 4 µL of GolgiStop for every 6 mL of cell culture and mix thoroughly. It is recommended that GolgiStop not be kept in cell culture for longer than 12 hours.
Note: It is recommended that kinetic studies be performed to determine the optimal incubation time for each experimental system.
2. Procedure for Using GolgiPlug Protein Transport Inhibitor (contains Brefeldin A)
GolgiPlug contains DMSO which is a solid at 4°C. Be sure to thaw at room temperature prior to use. Add 1 µL of GolgiPlug for every 1 mL of cell culture and mix thoroughly. It is recommended that GolgiPlug not be kept in cell culture for longer than 12 hours.
Note: It is recommended that kinetic studies be performed to determine the optimal incubation time for each experimental system. B. Protocol: Multicolor Staining for Cell Surface Antigens and
Intracellular Cytokines 1. Harvest Cells
Viable cell populations can be prepared from in vivo-stimulated tissues or from in vitro stimulatory cultures treated with a protein transport inhibitor. The cells should be spun down out of the medium containing GolgiStop or GolgiPlug, then resuspended in staining media, counted and transferred to plastic tubes or microwell plates for immunofluorescent staining. Cells should be protected from light throughout staining and storage.
2. Block Fc Receptors
Reagents that block Fc receptors may be useful for reducing nonspecific immunofluorescent staining.
a. In the mouse system, purified 2.4G2 antibody, specific for FcγII/ III receptors (Fc Block), can be used to block nonspecific staining by fluorochrome-conjugated antibodies which is mediated by Fc receptors. To block mouse Fc receptors with Fc Block, preincubate cell suspension with 1 µg Fc Block/106 cells in 100 µL of Staining Buffer for 15 minutes at 4°C. The cells can then be washed and stained with a fluorochrome-conjugated antibody specific for a cell surface antigen of interest which should be diluted appropriately in Staining Buffer.
b. Fc receptors on human cells can be pre-blocked by incubating cells with 10% normal human serum or an excess of irrelevant purified Ig from the same species and with the same isotype as the antibodies used for immunofluorescent staining. 3. Stain Cell Surface Antigens
a. Stain ~106 cells in 50 µL of Staining Buffer with the appropriate amount of a fluorochrome-conjugated monoclonal antibody specific for a cell surface antigen such as CD3, CD4, CD8, CD14, or CD19 (30 minutes, 4°C). Note: Multicolor staining of different cell surface antigens can be done at this time to provide controls for setting proper compensation of the brightest fluorescent signals. Some antibodies which recognize cell surface markers may not bind to fixed/denatured antigen. For this reason, it is recommended that the staining of cell surface antigens be done with live, unfixed cells PRIOR to fixation/ permeabilization and staining of intracellular cytokines. Altering the procedure such that cells are fixed prior to staining of cell surface antigens requires that suitable antibody clones be empirically identified.
b. Wash cells 2 times with Staining Buffer (e.g., 250 µL/wash for microwell plates, 1 mL/wash for tubes) and pellet by centrifugation (250 × g).
4. Fix and Permeabilize Cells
a. Thoroughly resuspend cells and add 100 µL per well for microwell plates (or 250 µL for tubes) of Fixation/ Permeabilization solution for 20 minutes at 4°C.
Note: Cell aggregation can be avoided by vortexing prior to the addition of the Fixation/Permeabilization solution.
b. Wash cells two times in 1× Perm/Wash buffer (e.g., 1 mL/ wash for staining in tubes and 250 µL/wash final volume for staining in microwell plates) and pellet.
Note: Perm/Wash buffer must be maintained in washing steps to keep cells permeabilized. 5. Alternative Fixation and Permeabilization Protocol
Cells can be fixed and stored to continue the intracellular staining at a later time.
a. Fixation and Storage of Cells
1. Resuspend cells in 100 µL (or 1 mL/107 cells for bulk fixing) of a 4% paraformaldehyde solution at 4°C for 10-20
minutes.
2. Wash cells 2× in Staining Buffer.
3. Resuspend cells in Staining Buffer for storing cells at 4°C for up to 72 hrs or in 90% FCS/10% DMSO for storing at -80°C for longer periods of time.
b. Permeabilizing Fixed Cells
1. For frozen cells, wash 2× to remove DMSO. For cells at 4°C, pellet and remove staining buffer.
2. Resuspend cells in Perm/Wash buffer for 15 minutes.
3. Pellet by centrifugation.
4. Stain for intracellular cytokines.
6. Stain for Intracellular Cytokines
a. Thoroughly resuspend fixed/permeabilized cells in 50 µL of Perm/Wash buffer containing a pre-determined optimal concentration of a fluorochrome-conjugated anti-cytokine antibody or appropriate negative control. Incubate at 4°C for 30 minutes in the dark.
b. Wash cells 2 times with 1× Perm/Wash buffer (1 mL/wash for staining in tubes and 250 µL/wash final volume for staining in microwell plates) and resuspend in Staining Buffer prior to flow cytometric analysis. C. Flow Cytometric Analysis Set PMT voltage and compensation using cell surface staining controls.
Set quadrant markers based on isotype or blocking controls and unstained cells. Note: Frequencies of cytokine producing cells derived from activation of human PBMCs can vary widely for a particular cytokine, depending on the donor. Cryopreserved cells from one donor can be used for longitudinal studies. For proper flow cytometric analysis, cells stained by this method should be inspected by light microscopy and/or flow light scatter pattern to confirm that they are well dispersed. In order to make statistically significant population frequency measurements, sufficiently large sample sizes should be acquired during flow cytometric analysis. Bivariate dot plots or probability contour plots can be generated upon data reanalysis to display the frequencies of and patterns by which individual cells coexpress certain levels of cell surface antigen and intracellular cytokine proteins. D. Staining Controls 1. Positive Staining Controls
The Technical Data Sheets for fluorochrome-conjugated
anticytokine antibodies provide specific examples of in vitro culture
systems which can induce detectable frequencies of cytokine-producing
cells at specific time points. Cells stimulated by these methods can be
used as positive controls for experimental systems. Particularly important parameters for cell activation protocols include the use of protein transport inhibitors and the examination of multiple time points.
Published reports of immunohistochemical staining and ELISPOT
analysis can also provide useful information regarding different
experimental protocols for generating cytokine-producing cells. 2. Negative Staining Controls
The use of at least one of the following three controls is suggested to
discriminate specific staining from artifactual staining. A combination of
unstained cells and isotype control or blocking control is optimal.
Investigators can choose which staining control best meets their research needs. Intracellular cytokine staining techniques and the use of blocking controls are described in detail by C. Prussin and D. Metcalf. a. Isotype control
Stain with an isotype-matched control of irrelevant specificity.
1. Resuspend cell pellet in 50 µL of Perm/Wash buffer containing a concentration of the isotype control antibody
equal to that of the anticytokine antibody.
2. Incubate 30 minutes at 4°C.
3. Wash cells using the aforementioned procedure for intracellular staining.
b. Ligand blocking control
Pre-block anti-cytokine antibody with recombinant cytokine.
1. preincubate fluorochrome-labeled antibodies with cytokine diluted to the appropriate concentration in at
least 50 µL of Perm/Wash buffer at 4°C for 30 minutes.
2. Resuspend fixed/permeabilized cells in 50 µL of preblocked labeled anticytokine antibody (in Perm/Wash buffer) and incubate 30 minutes at 4°C.
3. Wash cells using the aforementioned procedure for intracellular staining.
c. Unlabeled antibody blocking control preincubate cells with unlabeled antibody.
1. Resuspend fixed/permeabilized cells in 25 µL Perm/Wash buffer containing unlabeled anti-cytokine
antibody diluted to the appropriate concentration, and incubate 30 minutes at 4°C.
2. After incubation, add fluorochrome-labeled anti-cytokine antibody at optimal concentration in 25 µL Perm/Wash buffer (50 µL final volume) and incubate 30 minutes at 4°C.
3. Wash cells using the aforementioned procedure for intracellular staining.
Staining Buffer
Dulbecco's PBS (DPBS) without Mg2+ or Ca2+
1% heat-inactivated FCS
0.09% (w/v) sodium azide
Adjust buffer pH to 7.4 - 7.6, filter (0.2 µm pore membrane), and store at 4°C

For research use only. Not intended for any clinical use. No products from Creative Biolabs may be resold, modified for resale or used to manufacture commercial products without prior written approval from Creative Biolabs.

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Tel: 1-631-871-5806
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Email:

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