BD FastImmune™ CD107a (H4A3)

1 Becton, Dickinson and CompanyBD Biosciences2350 Qume DriveSan Jose, CA 95131 Research Use Only. Not for use in diagnostic or therapeutic procedures. Monoclonal Antibodies Detecting Human AntigensBD FastImmune CD107a (H4A3)FormCatalog numberAPC641581 Product availability varies by region. Contact BD Biosciences Customer Support or your local sales representative for APPLICATIONSThe BD FastImmune CD107a APC reagent is designed for the detection of degranulating T lymphocytes in activated whole blood and is intended for research use only. Applications include studies of cytotoxic CD8+ T- cell responses to viral and tumor antigens,1-6 correlation of T-cell cytolytic potential and cytokine expression,1 and live-cell sorting of functional antigen-specific CD8+ T The CD107a detection assay can also be used as an alternative to 51Cr release ,2,4 DESCRIPTIONS pecificityEach vial supplies sufficient reagents for 50 stimulated samples and 50 unstimulated control samples.

2 In performing the assay, mL of whole blood is stimulated with antigen (not included) in the presence of two secretion inhibitors, monensin and brefeldin A (BFA), and CD107a antibody. For the unstimulated control, mL of blood is treated with the secretion inhibitors and CD107a , but not antigen. Both blood samples are then stained for IFN- or other cytokine(s), as well as the gating markers CD3 and CD8 (not included).NOTEIf you are using specific antigen as the stimulus, you should activate an additional mL of blood with a superantigen, such as staphylococcal enterotoxin B (SEB). This sample is used as a positive activation control and simplifies technique detects cytolytic activity of CD8+ T cells by measuring degranulation, a prerequisite for Degranulating cells are identified by their surface expression of CD107a , which is a lysosomal associated membrane protein (LAMP-1) residing in cytolytic granule membranes located within the ,7 The marker is mobilized to the cell surface following activation-induced granule ,8,9 Because of their parallel kinetics, CD107a and intracellular cytokines can be assessed at the same time in short-term activated blood blood is stimulated with antigenic peptides or superantigens for 4 to 6 hours in the presence of the secretion inhibitors monensin and BFA.

3 As a result of degranulation, CD107a is expressed on the cell surface transiently and is rapidly re-internalized via the endocytic Therefore, CD107a detection is maximized by antibody staining during cell stimulation and by the addition of monensin (to prevent acidification and subsequent degradation of endocytosed CD107a antibody complexes). BFA is required for optimal cytokine activation, EDTA is added to remove adherent cells from the activation vessel, followed by the lysis of erythrocytes and fixation of leucocytes using BD FACS lysing solution. Cells are then washed and permeabilized using BD FACS permeabilizing solution 2. Cells are washed again and then stained for other surface and intracellular markers. Finally, cells are washed and fixed for analysis on a flow FastImmune CD107a APC, clone H4A3, is composed of mouse IgG1 heavy chains and kappa light 2 SPECIMEN COLLECTION AND PREPARATIONB lood should be collected in sodium heparin because other anticoagulants severely compromise the functional capacity of lymphocytes.

4 Blood should be stored at room temperature to avoid platelet activation prior to processing but should be processed within 8 hours of collection. Antigen-presenting cell function is compromised with longer storage times, and loss of function can be compounded by AND MATERIALS REQUIRED BUT NOT PROVIDED Heparinized whole blood Activation agent This kit is optimized for activation by a superantigen such as SEB. BD FastImmune brefeldin A (Catalog No. 347688)Store in aliquots at 20 C. BD FastImmune brefeldin A also contains dimethyl sulfoxide (DMSO). BD GolgiStop protein transport inhibitor (containing monensin, Catalog No. 554724)Store at 2 C 8 C. See the product insert for any warnings. BD FastImmune EDTA solution (Catalog No. 347689) BD FACS lysing solution (10X) (Catalog No. 349202)For dilution instructions and warnings, see the product insert. BD FACS permeabilizing solution 2 (Catalog No.)

5 340973 (25 mL) or 347692 (10 mL)For dilution instructions and warnings, see the product insert. Wash bufferFirst prepare stock solutions of 5% bovine serum albumin (BSA) in 1X phosphate-buffered saline (PBS) (filter sterilize) and 10% sodium azide (NaN3) in 1X PBS. Then prepare 500 mL of wash buffer by adding 50 mL of 5% BSA stock solution and 5 mL of 10% NaN3 stock solution to 445 mL of 1X sterile PBS. This represents final concentrations of BSA and NaN3 in PBS. Store at 4 C. 1% paraformaldehyde solution prepared in PBS containing sodium azideStore at 2 C 8 C in amber glass for up to 1 week. See the product insert for warnings. 15-mL polypropylene tubes (Catalog No. 352096) 5-mL polystyrene tubes (Catalog No. 352058) Vortex mixer Micropipettor with tips 37 C water bath or incubator Centrifuge BD FACS brand flow cytometerSee the appropriate cytometer user s guide for information.

6 BD Calibrite 3 beads (Catalog No. 340486) and BD Calibrite APC beads (Catalog No. 340487)See the BD Calibrite beads product insert for instructions . BD FACSComp software, version or later, for cytometer setup BD FACSDiva , BD CellQuest Pro, or BD CellQuest software for acquisition and analysis, or BD Paint-A-Gate Pro software for analysisSee the appropriate software user s guide for detailed 323-8946-02BD FACS 7-color setup beads (Catalog No. 335775)See the BD FACS 7-color beads product insert for the staining for CD107a is carried out during cell activation in this assay, CD107a APC reagent is formulated with low sodium azide concentration to minimize the possible compromising of the functional capacity of lymphocytes. Use sterile PBS for dilution and take care to avoid microbial Dilute an aliquot each of BFA and BD GolgiStop protein transport inhibitor 1:10 with sterile Label two 15-mL polypropylene tubes as follows.

7 Tube 1: Activated Tube 2: Unstimulated3. Add the following to the Activated tube. mL of heparinized whole blood antigen at titer (or other activation agent) 5 L of CD107a APC 5 L each of the diluted BFA and diluted BD GolgiStop protein transport inhibitor4. Add the following to the Unstimulated tube. mL of heparinized whole blood 5 L of CD107a APC 5 L each of the diluted BFA and diluted BD GolgiStop protein transport inhibitor5. Vortex each tube gently and incubate for 4 to 6 hours at 37 you are using a specific antigen for the activation agent, you should activate an additional mL of blood with a strong activation agent such as SEB (final concentration of 1 g/mL of blood). This tube is used as a positive control and simplifies Add 50 L of EDTA solution in PBS to each Vortex vigorously and incubate for 15 minutes in the dark at room temperature. 8. Vortex again on high setting for 10 cells are to be stained fresh, proceed with the next section, Preparing fresh cells.

8 If cells are to be frozen for later staining, go to Preparing frozen fresh cells1. Label two 5-mL polystyrene tubes as follows. Tube 1: Activated Sample (AS) Tube 2: Unstimulated Sample (US)2. Aliquot 200 L of activated blood into the AS Aliquot 200 L of unstimulated blood into the US Add 2 mL of 1X BD FACS lysing solution to each the 10X solution 1:10 with DI water before Mix gently and incubate for 10 minutes in the dark at room Add 1 mL of wash buffer to each Centrifuge at 500 x g for 5 minutes at room the to Permeabilizing and staining the cells (page 4).23-8946-02 Page 4 Preparing frozen cells1. Add 5 mL of 1X BD FACS lysing solution (dilute the 10X solution 1:10 with DI water before use) to each activated and unstimulated mL whole blood sample. 2. Vortex and incubate for 10 minutes in the dark at room temperature, and immediately place the tubes in a freezer at 80 C.

9 3. At the time of staining, thaw cells briefly in a 37 C water Add 7 mL of wash buffer, and centrifuge at 500 xg for 10 minutes at room Decant the supernatant and resuspend the pellet in mL of wash buffer. 6. When ready to stain, label two 5 mL polystyrene tubes and aliquot 200 L of blood as described in steps 1 through 3 of the previous section, Preparing fresh to the next section, Permeabilizing and staining the and staining the cells1. Add 1 mL of 1X BD FACS permeabilizing solution 2 to each the 10X solution 1:10 with DI water before Vortex to resuspend the pellet, and incubate for 10 minutes in the dark at room Add 2 mL of wash buffer to each tube, and centrifuge at 500 x g for 5 minutes at room the and permeabilized cells are more buoyant than live cells and require higher centrifugal force to pellet. Therefore we recommend that you decant to remove the supernatant instead of performing the typical Wash by adding 2 mL of wash buffer to each tube and centrifuging at 500 x g for 5 minutes at room the Add appropriate gating and intracellular cytokine reagents to the AS and US for 60 minutes in the dark at room temperature.

10 6. Add 2 mL of wash buffer to each tube, and centrifuge at 500 x g for 5 minutes at room the Wash one more time by adding 2 mL of wash buffer to each tube and centrifuging at 500 x g for 5 minutes at room the supernatant. 8. Add 300 L of 1% paraformaldehyde in PBS to each Vortex to resuspend the pellet, and store at 4 C in the dark before flow cytometry analysis. Analyze within 24 ACQUISITION AND ANALYSISA nalyze on a BD FACS brand flow cytometer with laser excitation at 488 nm and 635 nm, such as the BD FACSC anto II, the BD FACSC anto , the BD FACSC alibur , or the BD LSR II flow cytometer. For cytometer setup instructions , see the appropriate instrument user s guide. For BD FACSC anto II or BD FACSC anto cytometer setup, use BD FACS 7-color setup beads. See the product insert for instructions . For BD FACSC alibur cytometer setup, use BD Calibrite beads and appropriate software.