Human CD4+T Cell Care Manual
INSTRUCTION MANUALZBM0067.04

SHIPPING CONDITIONSHuman CD4+T Cells, cryopreserved

Cryopreserved human CD4+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 CD4+T cells are to be stored in vapor phase
nitrogen (-150C to -190C).
Lymphocyte Medium: Media: Short Term ( 30 days from ship date)
4°C6 months-20°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(919) 547-0692(919) 547-0693
1-866-ADIPOSE 1-(866)-234-7673
information@zenbio.com
http://www.zenbio.com

THIS MANUAL IS SUITABLE FOR USE WITH THE FOLLOWING PRODUCTS:
SER-CD4+T

Cryopreserved CD4+T Cells from Normal Human Peripheral Blood

SER-PBCD4+TH-N-F

Cryopreserved Naive CD4+ TH Cells from Normal Peripheral Blood

SER-CBCD4+T

Cryopreserved CD4+T Cells from Umbilical Cord Blood

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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
T Cells have a key function in the adaptive immune system. They can either promote growthand differentiation of other immune cells or show suppressive function and down-regulate immune
reactions.
The CD4+ T Cells are produced using an indirect magnetic labeling system for the isolation ofuntouched CD4+ T helper cells from human PBMCs at the Zen-bio facility. Non-CD4+ T cells, i.e., CD8+
T cells, B cells, NK cells, dendritic cells, monocytes, granulocytes, and erythroid cells, are screened for
CD8, CD14, CD15(16) CD19, CD56, and glycophorin A. CD4+ T lymphocyte cells are isolated fromnormal peripheral blood using negative immunomagnetic selection directed against the CD4 surface
antigen. Negative selection is preferred with immune cell isolations to avoid activation of the cells by theantibody.
CD4+ T lymphocytes play a central role in regulating the cell mediated immune response toinfection. These cells are often known as "helper" T cells, as they act on other cells of the immune
system to promote various aspects of the immune response, including immunoglobulin isotype
switching and affinity maturation of the antibody response, macrophage activation, and enhancedactivity of natural killer (NK) cells and cytotoxic T cells (CTL).
CD4+ T cells act by releasing cytokines in response to antigenic stimulation. Cytokines aresoluble intercellular messenger molecules, which interact with specific receptor molecules on their"target" cells. The release of cytokines allows cells of different types to "talk" to each other in the ongoing immune response. One of the major effector functions of CD4+ T cells is in the activation ofmacrophages. Macrophage activation plays an important role in enhancing bacterial killing at sites ofinfection. Applications for CD4+T cells include: drug testing and drug discovery, assessing immune
response, toxicology, genetic studies, inflammation, and autoimmune diseases.
ZenBio, Inc. CD4+ cells are isolated from anticoagulated blood collected in the United Statesfrom a volunteer adult donor who has signed an IRB or FDA validated donor consent form thatspecifically lists both the intended uses for the donation for non-clinical research and confirms theprocedures for processing the samples are Standard Operating Procedure (SOP) managed GLP
protocols in compliance with all legal and ethical regulations.

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QUALITY CONTROL
Quality control tests are performed for each lot of CD4+T cells. The cells are characterized bytheir surface markers via flow cytometry. Population distributions are expressed as percentage positive
are presented on the certificate of analysis for each lot of cells. Cells have a guaranteed purity of >95%
and a 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 CD4+T Cells from Normal Human Peripheral Blood
Catalog #: SER-CD4+T
Frozen vial containing 5.0 million cells/vial
 Cryopreserved Naive CD4+ TH Cells from Normal Peripheral Blood
Catalog #: SER-PBCD4+TH-N-F
Frozen vial containing 1.0 million cells/vial
 Cryopreserved CD4+T Cells from Umbilical Cord Blood
Catalog #: SER-CBCD4+T
Frozen vial containing 5.0 million cells/vial

LYMPHOCYTE MEDIUM COMPOSITION
Recommended product for thawing cells.
Cat# LYMPH-1 (100ml); LYMPH-1-50 (50ml)
Medium Composition
RPMI 1640
L-GlutamineFetal Bovine Serum
DNAse I
Penicillin
Streptomycin

Expiration Dates
If placed at 4C upon arrival, the media isstable until the expiration date on the bottlelabel which is 30 days from the shipping date.
If stored at -20C upon arrival, the media
is stable for 6 months. Add fresh antibiotics
when you are ready to use. The media will
expire 30 days after the thaw date.

Amphotericin B
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THAWING CRYOPRESERVED CD4+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.

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 is
NOT 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.

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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, Hepatitis B, Hepatitis C, and syphilis. However,since we cannot test all pathogens, please treat the culture as a potentially 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. All
our 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 buta medium designed to successfully thaw blood derived cells.

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

株式会社ケー・エー・シー

human cells.

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試薬事業部〒661-0978
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