Monoclonal Antibodies Detecting Human Antigens

RESEARCH APPLICATIONS

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BD FastImmune™ CD107a (H4A3) Form APC

Catalog number 641581

Product availability varies by region. Contact BD Biosciences Customer Support or your local sales representative for information.

The 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 livecell sorting of functional antigen-specific CD8+ T cells.2 The CD107a detection assay can also be used as an alternative to 51Cr release assays.1,2,4

DESCRIPTION Specificity

Each vial supplies sufficient reagents for 50 stimulated samples and 50 unstimulated control samples. In performing the assay, 0.5 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, 0.5 mL of blood is treated with the secretion inhibitors and CD107a, but not antigen. Both blood samples are then stained for IFN-γ or other cytokine(s), as well as the gating markers CD3 and CD8 (not included). NOTE If you are using specific antigen as the stimulus, you should activate an additional 0.5 mL of blood with a superantigen, such as staphylococcal enterotoxin B (SEB). This sample is used as a positive activation control and simplifies gating. This technique detects cytolytic activity of CD8+ T cells by measuring degranulation, a prerequisite for cytolysis.1 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 cytoplasm.1,7 The marker is mobilized to the cell surface following activation-induced granule exocytosis.1,8,9 Because of their parallel kinetics, CD107a and intracellular cytokines can be assessed at the same time in short-term–activated blood samples. Whole blood is stimulated with antigenic peptides or superantigens for 4 to 6 hours in the presence of the secretion inhibitors monensin and BFA. As a result of degranulation, CD107a is expressed on the cell surface transiently and is rapidly re-internalized via the endocytic pathway.9 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 expression. After 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 cytometer.

Composition

BD FastImmune CD107a APC, clone H4A3, is composed of mouse IgG1 heavy chains and kappa light chains. For Research Use Only. Not for use in diagnostic or therapeutic procedures.

Becton, Dickinson and Company BD Biosciences 2350 Qume Drive San Jose, CA 95131 USA

bdbiosciences.com [email protected]

11/2014

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SPECIMEN COLLECTION AND PREPARATION

Blood should be collected in sodium heparin because other anticoagulants severely compromise the functional capacity of lymphocytes. 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 shipping.

REAGENTS AND MATERIALS REQUIRED BUT NOT PROVIDED

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Heparinized whole blood

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Activation agent This kit is optimized for activation by a superantigen such as SEB.

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BD FastImmune™ brefeldin A (Catalog No. 347688) Store in aliquots at –20°C. BD FastImmune brefeldin A also contains dimethyl sulfoxide (DMSO).

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BD GolgiStop™ protein transport inhibitor (containing monensin, Catalog No. 554724) Store at 2°C–8°C. See the product insert for any warnings.

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BD FastImmune™ EDTA solution (Catalog No. 347689)

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BD FACS lysing solution (10X) (Catalog No. 349202) For dilution instructions and warnings, see the product insert.

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BD FACS permeabilizing solution 2 (Catalog No. 340973 (25 mL) or 347692 (10 mL) For dilution instructions and warnings, see the product insert.

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Wash buffer First prepare stock solutions of 5% bovine serum albumin (BSA) in 1X phosphatebuffered 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 0.5% BSA and 0.1% NaN3 in PBS. Store at 4°C.

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1% paraformaldehyde solution prepared in PBS containing 0.1% sodium azide Store at 2°C–8°C in amber glass for up to 1 week. See the product insert for warnings.

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15-mL polypropylene tubes (Catalog No. 352096)

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5-mL polystyrene tubes (Catalog No. 352058)

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Vortex mixer

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Micropipettor with tips

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37°C water bath or incubator

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Centrifuge

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BD FACS™ brand flow cytometer See the appropriate cytometer user’s guide for information.

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BD Calibrite™ 3 beads (Catalog No. 340486) and BD Calibrite™ APC beads (Catalog No. 340487) See the BD Calibrite beads product insert for instructions.

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BD FACSComp™ software, version 4.2 or later, for cytometer setup

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BD FACSDiva™, BD CellQuest™ Pro, or BD CellQuest™ software for acquisition and analysis, or BD Paint-A-Gate™ Pro software for analysis See the appropriate software user’s guide for detailed information.

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BD FACS™ 7-color setup beads (Catalog No. 335775) See the BD FACS 7-color beads product insert for instructions. PROCEDURE

Because 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 contamination. 1. Dilute an aliquot each of BFA and BD GolgiStop protein transport inhibitor 1:10 with sterile PBS. 2. Label two 15-mL polypropylene tubes as follows. •

Tube 1: Activated

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Tube 2: Unstimulated

3. Add the following to the Activated tube. •

0.5 mL of heparinized whole blood

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antigen at titer (or other activation agent)

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5 µL of CD107a APC

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5 µL each of the diluted BFA and diluted BD GolgiStop protein transport inhibitor

4. Add the following to the Unstimulated tube. •

0.5 mL of heparinized whole blood

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5 µL of CD107a APC

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5 µL each of the diluted BFA and diluted BD GolgiStop protein transport inhibitor

5. Vortex each tube gently and incubate for 4 to 6 hours at 37°C. NOTE If you are using a specific antigen for the activation agent, you should activate an additional 0.5 mL of blood with a strong activation agent such as SEB (final concentration of 1 µg/mL of blood). This tube is used as a positive control and simplifies gating. 6. Add 50 µL of EDTA solution in PBS to each tube. 7. Vortex vigorously and incubate for 15 minutes in the dark at room temperature. 8. Vortex again on high setting for 10 seconds. If cells are to be stained fresh, proceed with the next section, Preparing fresh cells. If cells are to be frozen for later staining, go to Preparing frozen cells. Preparing fresh cells

1. Label two 5-mL polystyrene tubes as follows. •

Tube 1: Activated Sample (AS)

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Tube 2: Unstimulated Sample (US)

2. Aliquot 200 µL of activated blood into the AS tube. 3. Aliquot 200 µL of unstimulated blood into the US tube. 4. Add 2 mL of 1X BD FACS lysing solution to each tube. Dilute the 10X solution 1:10 with DI water before use. 5. Mix gently and incubate for 10 minutes in the dark at room temperature. 6. Add 1 mL of wash buffer to each tube. 7. Centrifuge at 500 x g for 5 minutes at room temperature. Decant the supernatant. Proceed to Permeabilizing and staining the cells (page 4). Page 3

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Preparing frozen cells

1. 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 0.5 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. 3. At the time of staining, thaw cells briefly in a 37°C water bath. 4. Add 7 mL of wash buffer, and centrifuge at 500 x g for 10 minutes at room temperature. 5. Decant the supernatant and resuspend the pellet in 0.5 mL of wash buffer. 6. When ready to stain, label two 5 mL polystyrene tubes and aliquot 200 µL of blood as described in steps 1 through 3 of the previous section, Preparing fresh cells. Proceed to the next section, Permeabilizing and staining the cells.

Permeabilizing and staining the cells

1. Add 1 mL of 1X BD FACS permeabilizing solution 2 to each tube. Dilute the 10X solution 1:10 with DI water before use. 2. Vortex to resuspend the pellet, and incubate for 10 minutes in the dark at room temperature. 3. Add 2 mL of wash buffer to each tube, and centrifuge at 500 x g for 5 minutes at room temperature. Decant the supernatant. NOTE Fixed 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 aspiration. 4. Wash by adding 2 mL of wash buffer to each tube and centrifuging at 500 x g for 5 minutes at room temperature. Decant the supernatant. 5. Add appropriate gating and intracellular cytokine reagents to the AS and US tubes. Incubate for 60 minutes in the dark at room temperature. 6. Add 2 mL of wash buffer to each tube, and centrifuge at 500 x g for 5 minutes at room temperature. Decant the supernatant. 7. 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 temperature. Decant the supernatant. 8. Add 300 µL of 1% paraformaldehyde in PBS to each tube. 9. Vortex to resuspend the pellet, and store at 4°C in the dark before flow cytometry analysis. Analyze within 24 hours.

DATA ACQUISITION AND ANALYSIS

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Analyze on a BD FACS brand flow cytometer with laser excitation at 488 nm and 635 nm, such as the BD FACSCanto™ II, the BD FACSCanto™, the BD FACSCalibur™, or the BD™ LSR II flow cytometer. For cytometer setup instructions, see the appropriate instrument user’s guide. •

For BD FACSCanto II or BD FACSCanto cytometer setup, use BD FACS 7-color setup beads. See the product insert for instructions.

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For BD FACSCalibur cytometer setup, use BD Calibrite beads and appropriate software. Use the 4-color lyse/no-wash (LNW) setting. Minor adjustment of PMT settings and compensation might be required. See the instructions for use for the beads and the software. Page 4

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For BD LSR II cytometer setup, see the application note BD™ Digital Flow Cytometer. Visit our website (bdbiosciences.com) or contact your local BD representative.

See Figure 1 and Figure 2 on page 5 for representative data from experiments performed on normal heparinized whole blood, activated with SEB, stained with CD3 PE and CD8 PerCP-Cy™5.5 as gating reagents, IFN-γ FITC as detecting intracellular cytokine, and analyzed on a BD FACSCalibur flow cytometer. 1. Acquire data with the appropriate software, using a forward scatter (FSC) threshold. •

Collect at least 30,000 preferably 40,000 CD3+ lymphocytes.

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During acquisition, set up an FSC vs SSC dot plot (Figure 1).

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Gate on the lymphocytes (R1).

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In addition, create a CD3 vs SSC dot plot with R1-gated lymphocytes, and draw a region around CD3+ cells (R2).

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Collect at least 30,000 events, preferably 40,000 events, that fall in R1 and R2.

Figure 1 Gating strategy R1-gated

SSC-H 200 400 600 800 1000

SSC-H 200 400 600 800 1000

ungated

R2

0

0

R1

0

200 400 600 800 1000 FSC-H

100

101

102 103 CD3 PE

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2. Create a CD3 vs CD8 dot plot to obtain double-positive cells (R3). Display data as cytokine vs CD107a dot plots to determine CD107a and cytokine expression (Figure 2). 3. Analyze data using BD FACSDiva, BD CellQuest Pro, BD CellQuest, or BD Paint-A-Gate Pro software. Figure 2 Unstimulated and SEB-activated samples, R1- and R3-gated

101 101

102 CD3 PE

103

104

100

R1- and R3-gated SEB-activated

CD107a APC 103 101 102

CD107a APC 102 103

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R1- and R3-gated unstimulated

102 103 101 Anti–IFN-γ FITC

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100

100

R3

100

100

CD8 PerCP-Cy5.5 101 102 103

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R1-gated

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101 102 103 Anti–IFN-γ FITC

Gated Events: 11433

Gated Events: 10406

Quad %Gated UL 0.39 UR 0.03 LL 99.20 LR 0.38

Quad %Gated UL 4.02 UR 17.35 LL 72.72 LR 5.92

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4. To obtain statistics, draw a quadrant region based on the unstimulated sample and apply the region to the activated sample files (Figure 2). The %Gated statistic gives frequency of CD107a and cytokine-producing CD3+CD8+ cells.

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For CD107a expression: obtain the sum of %Gated statistics from the UR and UL region.

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For cytokine expression: obtain the sum of %Gated statistics from the UR and LR region. 23-8946-02

Calculating the specific response

The specific response of cells to any stimulus is obtained by subtracting the percent positive events in the unstimulated sample from the percent positive events in the activated sample.

LIMITATIONS

Specific responses will vary by donor and by cytokine.

HANDLING AND STORAGE

Store vials at 2°C–8°C. Conjugated forms should not be frozen. Protect from exposure to light. Each reagent is stable until the expiration date shown on the bottle label when stored as directed. Alteration in the appearance of any reagent, such as precipitation or discoloration, indicates instability or deterioration. In such cases, do not use the reagent.

WARNING

All biological specimens and materials coming in contact with them are considered biohazards. Handle as if capable of transmitting infection10,11 and dispose of with proper precautions in accordance with federal, state, and local regulations. Never pipette by mouth. Wear suitable protective clothing, eyewear, and gloves.

WARRANTY

Unless otherwise indicated in any applicable BD general conditions of sale for non-US customers, the following warranty applies to the purchase of these products. THE PRODUCTS SOLD HEREUNDER ARE WARRANTED ONLY TO CONFORM TO THE QUANTITY AND CONTENTS STATED ON THE LABEL OR IN THE PRODUCT LABELING AT THE TIME OF DELIVERY TO THE CUSTOMER. BD DISCLAIMS HEREBY ALL OTHER WARRANTIES, EXPRESSED OR IMPLIED, INCLUDING WARRANTIES OF MERCHANTABILITY AND FITNESS FOR ANY PARTICULAR PURPOSE AND NONINFRINGEMENT. BD’S SOLE LIABILITY IS LIMITED TO EITHER REPLACEMENT OF THE PRODUCTS OR REFUND OF THE PURCHASE PRICE. BD IS NOT LIABLE FOR PROPERTY DAMAGE OR ANY INCIDENTAL OR CONSEQUENTIAL DAMAGES, INCLUDING PERSONAL INJURY, OR ECONOMIC LOSS, CAUSED BY THE PRODUCT.

REFERENCES

1.

Betts MR, Brenchley JM, Price DA, et al. Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J Immunol Meth. 2003;281:65-78.

2.

Rubio V, Stuge TB, Singh N, et al. Ex vivo identification, isolation and analysis of tumor-cytolytic T cells. Nat Med. 2003;9:1377-1382.

3.

Betts MR, Price DA, Brenchley JM, et al. The functional profile of primary human antiviral CD8+ T cell effector activity is dictated by cognate peptide concentration. J Immunol. 2004;172:6407-6417.

4.

Mittendorf EA, Storrer CE, Shriver CD, Ponniah S, Peoples GE. Evaluation of the CD107 cytotoxicity assay for the detection of cytolytic CD8+ cells recognizing HER2/neu vaccine peptides. Breast Cancer Res Treat. 2005;92:85-93.

5.

Nagorsen D, Scheibenbogen C, Thiel E, Keilholz U. Immunological monitoring of cancer vaccine therapy. Expert Opin Biol Ther. 2004;4:1677-1684.

6.

Jones N, Eggena M, Baker C, et al. Presence of distinct subsets of cytolytic CD8+ T cells in chronic HIV infection. AIDS Res Hum Retroviruses. 2006;22:1007-1013.

7.

Betts MR, Koup RA. Detection of T-cell degranulation: CD107a and b. Methods Cell Biol. 2004;75:487-512.

8.

Peters PJ, Borst J, Oorschot V, et al. Cytotoxic T lymphocyte granules are secretory lysosomes, containing both perforin and granzymes. J Exp Med. 1991;173:1099-1109.

9.

Fukuda M. Lysosomal membrane glycoproteins: structure, biosynthesis, and intracellular trafficking. J Biol Chem. 1991;266:21327-21330.

10. Protection of Laboratory Workers from Occupationally Acquired Infections; Approved Guideline —Third Edition. Wayne, PA: Clinical and Laboratory Standards Institute; 2005. CLSI document M29-A3. 11. Centers for Disease Control. Perspectives in disease prevention and health promotion update: universal precautions for prevention of transmission of human immunodeficiency virus, hepatitis B virus, and other bloodborne pathogens in health-care settings. MMWR. 1988;37:377-388.

PATENTS AND TRADEMARKS

Cy™ is a trademark of GE Healthcare. This product is subject to proprietary rights of GE Healthcare and Carnegie Mellon University, and is made and sold under license from GE Healthcare. This product is licensed for sale only for research. It is not licensed for any other use. If you require a commercial license to use this product and do not have one, return this material, unopened, to BD Biosciences, 2350 Qume Drive, San Jose, CA 95131, and any money paid for the material will be refunded. BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. © 2014 BD

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