Human CD3+T Care Manual
INSTRUCTION MANUALZBM0086.01

SHIPPING CONDITIONSHuman CD3+T, cryopreserved

Cryopreserved human CD3+T cells are shipped on dry ice and should be stored in liquid
nitrogen vapor phase immediately upon arrival. Orders are delivered via overnight courier.
Must be processed immediately upon shipment receipt.

STORAGE CONDITIONSCryopreserved cells: Vials of frozen CD3+T cells are to be stored in vapor phase
nitrogen (-150C to -190C).
Lymphocyte Medium: Store 2-8C.
All Zen-Bio Inc products are for research use only. Not approved for human orveterinary use or for use in diagnostic or clinical procedures.

ORDERING INFORMATION AND TECHNICAL SERVICES
ZenBio, Inc.
3200 East NC-54 Suite 100
PO Box 13888
Research Triangle Park, NC 27709
U.S.A.
Telephone
Facsimile (FAX)
Toll free (continental US only)
Electronic mail (e-mail)World Wide Web

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1-866-ADIPOSE 1-(866)-234-7673
information@zenbio.com
http://www.zenbio.com

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LIMITED PRODUCT WARRANTYThis warranty limits our liability to replacement of this product. No other warranties of any kind,
expressed or implied, including without limitation implied warranties of merchantability or fitness for aparticular purpose, are provided by Zen-Bio, Inc. Zen-Bio, Inc. shall have no liability for any direct,
indirect, consequential, or incidental damages arising out of the use, the results of use, or the inability
to use this product.
Zen-Bio, Inc warrants its cells only if Zen-Bio media are used and the recommended protocols
are followed. Cryopreserved human blood cells are assured to be viable when thawed according toZen-Bio protocols.
Contact ZenBio, Inc. within no more than 24 hours after receipt of products for all claims
regarding shipment damage, incorrect ordering or other delivery issues. Delivery claims received after 7
days of receipt of products are not subject to replacement or refund.

PRECAUTIONSThis product is for research use only. It is not intended for human, veterinary, or in vitro
diagnostic use. Proper precautions and biological containment should be taken when handling cells ofhuman origin, due to their potential biohazardous nature. Always wear gloves and work behind aprotective screen when handling primary human cells. All media, supplements, and tissue culture wareused in this protocol should be sterile.
To comply with U.S. Food and Drug Administration (FDA) regulations, these products are not for
use in Clinical Diagnostic or Therapeutic Procedures.
By your acceptance of these products, you are acknowledging that these products will be:

1. Treated as potentially contaminated biological specimens even if accompanying serologicalreports are negative;

2. Handled by establishing or following appropriate safety control procedures to ensure the safetyof using these products.

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INTRODUCTION
CD3 is initially expressed in the cytoplasm of pro-thymocytes, the stem cells from which T-cells
arise in the thymus. The pro-thymocytes differentiate into common thymocytes, and then into medullary
thymocytes, and it is at this latter stage that CD3 antigen begins to migrate to the cell membrane. The
antigen is found bound to the membranes of all mature T-cells, and in virtually no other cell type,although it does appear to be present in small amounts in Purkinje cells. This high specificity, combinedwith the presence of CD3 at all stages of T-cell development, makes it a useful immunohistochemicalmarker for T-cells in tissue sections. The antigen remains present in almost all T-cell lymphomas andleukaemias, and can therefore be used to distinguish them from superficially similar B-cell and myeloid
neoplasms. Because CD3 is required for T-cell activation, drugs (often monoclonal antibodies) thattarget it are being investigated as immunosuppressant therapies (e.g., otelixizumab) for type 1 diabetes
and other autoimmune diseases.
CD3 is expressed on all T cells and CD56+ NKT cells, and is associated with the T cell
receptor. 70–80% of human peripheral blood lymphocytes and 65–85% of thymocytes are CD3+CD3+ T cells are isolated from normal peripheral blood using negative immunomagnetic
selection directed against the CD3 surface antigen. Negative selection is preferred with immune cellisolations to avoid activation of the cells by the antibody.

QUALITY CONTROL
Quality control tests are performed for each lot of CD3+T cells. The cells are characterized bytheir surface markers via flow cytometry. Population distributions expressed as percentage positive arepresented on the certificate of analysis for each lot of cells. Cells have a guaranteed purity of >95% anda viability >80%. In addition, all blood products have been tested for some common blood borne
pathogens and microbial contaminants.

MATERIALS PROVIDED FOR EACH CATALOG ITEM
 Cryopreserved CD3+T Cells from Normal Human Peripheral Blood
Catalog # SER-CD3-F
Frozen vial containing 5 million cells/vial

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LYMPHOCYTE MEDIUM COMPOSITION
Recommended product.
Cat# LYMPH-1 (100ml); LYMPH-1-50 (50ml)
RPMI 1640
L-Glutamine
Fetal Bovine Serum
DNAse I
Penicillin
Streptomycin
Amphotericin B

THAWING CRYOPRESERVED CD3+T cells
1. Warm the appropriate medium that will be used to thaw the cells to 37°C.
2. Rapidly thaw the vial of frozen cells in a 37°C water bath until just prior to complete thawing(slurry of residual ice should be present). Wipe the outside of the vial with 70% ethanol.

3. Aseptically transfer the cell suspension to a 50mL conical tube.
4. Rinse the vial with 1 mL of medium. Then slowly add drop wise to the cells in the 50 mL conicaltube while gently swirling the tube.

5. Slowly add medium drop wise to the 50 mL tube until the total volume reaches 25 ml.
6. Centrifuge the cell suspension at 400x g at room temperature for 10 minutes.
7. Carefully remove the supernatant and save in a second tube leaving 1 mL behind as not todisturb the pellet. Gently resuspend the cells up to a volume of 2 mL (2 mL per vial of product).
Count the number of cells. If count is lower than expected, centrifuge the wash that was saved
at a higher speed, count and combine if necessary.

8. Gently resuspend cells to desired concentration.

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FREQUENTLY ASKED QUESTIONS
1. Must I use your Lymphocyte Medium? Yes, we strongly recommend the use of ourLymphocyte Medium to thaw the cells as it will prevent clumping and maximize viability upon
thawing. If you are using a homemade formulation and not achieving success, please use ourLymphocyte Medium in a variety of convenient sizes to suit your needs (catalog # LYMPH-1,LYMPH-1-50).
2. Can I use your Lymphocyte Medium to culture my cells? No. Our Lymphocyte Medium isNOT a culture or a growth medium. It is a medium designed to successfully thaw blood derived
cells with high viability and less clumping of the subpopulations of cells that remain insuspension.
3. Do you test for pathogens? Which ones? Yes. Samples from each donor are tested via
PCR to confirm non-reactivity for HIV-1, HIV-2, HTLV I, HTLV II, syphilis, CMV, hepatitis B andhepatitis C. However, since we cannot test all pathogens, please treat the culture as apotentially infectious agent at Biosafety Level 2 or higher.
4. What donor information do I receive? The donor’s age, race, and gender are provided in thecertificate of analysis that accompanies each lot of cells.
5. Do you have any protocols for ways to use the cells? No. We do not provide any protocols
for the use of the peripheral blood mononuclear cells. The uses for this product are too varied toprovide a comprehensive protocol suitable for each experiment.
6. My cells have low viability and are clumping upon thawing. Is there a problem with mycells? We first eliminate any shipping or storage issues as a potential source of your issues. Allour cells are quality tested with a minimum viability greater than 80% upon thawing fromcryopreservation. We strongly suggest the use of our Lymphocyte Medium to thaw the cells as it
will prevent clumping and maximize viability upon thawing. If you are using a homemade
formulation and not achieving success, please use our Lymphocyte Medium (catalog # LYMPH1, LYMPH-1-50).
7. My cells are not attaching or proliferating. What is wrong? Nothing is wrong. We
recommend that you thaw and use the cells directly. The factors used to treat your cells will
depend on your research goal. Our Lymphocyte Medium is NOT a culture or growth medium but
a medium designed to successfully thaw blood derived cells.

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PATHOGEN TESTING
Samples from each donor are tested via PCR to confirm non-reactivity for HIV-1, HIV-2, HTLV I,HTLV II, syphilis, CMV, hepatitis B and hepatitis C. However, no known test can offer complete
assurance that the cells are pathogen free. Our products are tested and are free from mycoplasma
contamination. Proper precautions and biological containment should be taken when handling cells ofhuman origin, due to their potential biohazardous nature. All human based products should be handled
at a BSL-2 (Biosafety Level 2) or higher. Always wear gloves and work behind a protective screen
when handling primary human cells.

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