|Frequently asked questions
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Product Form Descriptions
Supernatant is serum-free hybridoma conditioned culture media containing bovine
serum albumin for protein stabilization (HB101 medium).
produced either by ultrafiltration through a 100 kD exclusion filter or by
dilution of bioreactor fluid. Concentrate is not a purified product but does contain 10 times the immunoglobulin
found in the supernatant. Therefore, 100 µl of concentrate contains the same amount of monoclonal antibody as 1 ml of
supernatant. Concentrate is recommended only for protocols requiring high antibody concentration in a small volume
(i.e., immunoprecipitation, microinjection or antibody conjugation). For routine staining or blotting protocols,
supernatant is recommended.
Bioreactor Supernatant is produced by sequestering hybridoma cells in a specialized tissue culture apparatus,
(bioreactor) with media that allows for the production of highly concentrated supernatant.
Ascites is the accumulation of excess fluid due to growth of hybridomas after
injection of the cells into the peritoneal space of a mouse. Ascites fluid will contain highly concentrated monoclonal
antibody along with other mouse proteins. The ascites product available from DSHB has been centrifuged and filter sterilized.
The DSHB no longer produces ascites fluid; only limited amounts remain available.
Datasheets are available at the website. Antibody concentration is provided on each tube label.
Frozen Cells are a 1mL aliquot containing approximately 5x106 to 1x107 cells. They are shipped on dry ice. When a customer receives them they are either thawed and put in culture immediately or placed in liquid nitrogen storage until they are needed.
Growing Cells are a live culture containing approximately 2x107 to 4x107 cells derived from a single frozen aliquot. We thaw the cells here, make sure they are healthy and growing well and then ship them in 65mL of media in a T25 flask. When the customer receives them they are immediately placed in an incubator for culture.
Frozen and growing hybridoma cells are shipping with full identification data and an outline of recommended protocols for culture and passage. Not all hybridoma cell lines are available.
To increase product stability, the antimicrobial agent ProClin 150 is now added to all DSHB products (supernatant, concentrate, biosupe, and ascites fluid).
||How does ProClin 150 work?
||ProClin kills microbes by inhibiting three key enzymes in the KREBS cycle resulting in the termination of respiration and protein synthesis.
||Will ProClin 150 reduce the activity of the monoclonal antibody?
||ProClin 150 was developed as a broad spectrum biocide specifically for affinity reagents, including antibodies, used in diagnostic laboratories.
||Does ProClin 150 interfere with horseradish peroxidase or alkaline phosphatase enzyme activity?
||ProClin does not inhibit HRP or Alk Phos enzyme activity immunohistochemical staining or western blots.
||Is ProClin 150 autofluorescent?
||ProClin 150 is not autofluorescent at the concentrations used.
||What safety measures do I need to use antibody solutions containing ProClin 150?
||The effective concentration of ProClin 150 is 15 ppm. Therefore, good laboratory practices are sufficient to limit exposure.
||How do I remove ProClin 150 from an antibody solution?
||Exhaustive dialysis may not be effective to remove ProClin 150 from an antibody solution. Purification of the antibody by affinity chromatography may be sufficient to remove residual ProClin 150. If your antibody protocol requires no ProClin such as immunostaining living cells for FACS sorting, contact the DSHB at 319-335-3826.
Although many cell products are maintained at 4°C for years without loss of activity, each antibody is different, and the shelf-life at 4°C is highly variable. To ensure retention of antibody activity, we recommend aliquotting the product into two parts: 1) a volume of antibody stored at 4°C to be used within two weeks and 2) the remaining product diluted with an equal volume of reagent grade glycerol and stored at -20°C.
Supernatant Use Recommendations
Although the optimal Ig concentration for an application varies for each monoclonal antibody and must be determined empirically, a good starting concentration for immunohistochemistry (IHC) is 2-5 µg/ml. For Western blots, the concentration is decreased by one order of magnitude (that is, 0.2-0.5 µg/ml).
Hybridoma Cell Culture
Frozen cells purchased from the DSHB should be thawed immediately or placed into the vapor phase of liquid nitrogen for storage. Thaw the cells in less than 2 minutes in a 37°C water bath. When the cells are just thawed, quickly transfer them to a tube containing 5ml of culture medium. Spin the cells at 170Xg for 5 minutes. Carefully draw off and discard the medium. Transfer cells to a T-75 flask containing 10 ml of complete culture medium. Flasks should be placed in an incubator at 37°C with 5.0% CO2. Maintain the culture by keeping the density between 5X105 and 1X106 cells/ml by adding culture medium over the course of several days. Split the culture into two T75 flasks, one for frozen aliquots and one for antibody supernatant.
Frozen cell aliquots can be made while the cells are in log phase growth and the density reaches approximately 5X105 and 1X106 cells/ml. Pellet the culture by spinning at 170Xg rpm for 5 minutes. Carefully draw off and discard the supernatant, resuspend the cell pellet in culture medium with cryoprotectant (6% DMSO), and transfer to the suspension to cryovials. Place the cryovials in the vapor phase of liquid nitrogen and freeze at a concentration of approximately 1X107 cells/ml. Normally, three 1 ml aliquots can be frozen from a 40ml culture.
Antibody supernatant is made by keeping the cell density of the culture between 5X105 and 1X106 cells/ml while adding serum-free medium over the course of several days. Split the culture into two flasks if a larger volume of supernatant is needed (200 ml is a normal production run). When half the desired volume of supernatant is reached, transfer the culture into a non-vented 490 cm2 roller bottle and double the volume by adding serum-free medium. The roller incubator should be set at 37°C and bottles should rotate at approximately 1 rpm. If a roller bottle setup is not available, the antibody production phase can be done in multiple static flasks with approximately the same combined surface area as the roller bottle. Allow 10 to 14 days before harvesting the supernatant. Hybridomas are not efficient in immunoglobulin secretion during log phase growth. Allow the culture to enter stationary phase and, eventually, death phase. Maximum immunoglobulin concentration is reached after 10-14 days when no more live cells remain. Spin the culture at 670Xg for 10 minutes. Retain the supernatant and filter sterilize. Discard the cells. For a typical production run of 200 ml of antibody supernatant, add 6 ml of 1M Tris-HCL pH 7.5 buffer to stabilize the pH. A volume of antibody can be stored at 4°C to be used within two weeks and the remaining product diluted with an equal volume of reagent grade glycerol and stored at -20°C.
For additional information contact us at 319-335-3826 or firstname.lastname@example.org.
US orders: To be sure your order is shipped the same week you order, please place your order by 11 AM Central time on Wednesday. If the order is correct and the items are available, we will do our best to ship for delivery that week.
International orders: Be sure to place your order by 11 AM US Central time on Thursday. If the order is correct and the items are available, we will do our best to ship the following Monday or Tuesday, depending on the country.
US shipping details
Shipping costs are estimates only; actual costs may differ. Shipping charge for domestic overnight delivery of cell products is $20.00 per shipment. Shipment of frozen cells and delivery to extended areas will be higher.
Freight terms are FOB Origin via DSHB-preferred carrier, pre-paid and added to the invoice. Purchase orders should indicate freight terms.
International shipping cost
For the majority of countries, international shipping cost will be $35. This is an estimate only; shipping cost to select countries can be as much as $80 to ship supernatant, concentrate, ascites fluid and growing cells. Estimated international shipment cost of frozen cells is $100-$150. Please note that some areas incur an additional extended delivery area fee. Requests to include special documents in shipments for customs clearance will incur an additional $10 fee at our discretion. Please contact us at email@example.com with any questions.
International shipping details
We ship internationally via FedEx, UPS, or DHL at ambient temperature (except in the case of frozen cells which ship on dry ice) and request refrigeration during any customs delays. *Please let us know when your
institution will be closed for holidays so we will be sure not to ship then.
International customs duties and taxes
It is the responsibility of the customer to make any arrangements necessary for prompt payment of customs duties assessed on an order shipped from the DSHB. We make every effort to provide the required paperwork for customs clearance. However if special circumstances exist, please advise us of any anticipated difficulty as soon as possible. Please provide us with your importer registration/identification number for clearance (i.e. EIN, SSN, VAT, GST, RFC, etc.) if applicable. We prepay the duties at these current rates for the following countries to avoid lengthy customs delays: Australia & New Zealand ($30), Italy ($36) and The Netherlands ($36).
Pre-payment of customs duties OPTION
To alleviate delivery delays resulting from the freight carrier demanding cash payment of customs duties upon delivery, when possible, we can pre-pay customs duties on international shipments. These fees are then included as a separate charge on the invoice you receive from the DSHB. To use this service option, you must request pre-payment of customs duties when placing your order with the DSHB. When requesting products from the DSHB, please include a customs duty estimate on your purchase order. We would appreciate your comments and suggestions regarding this matter.
Contact us at firstname.lastname@example.org or by fax at 1-319-335-2077 with any questions. Thank you.
Shipping to China
Because of the difficulty the DSHB has shipping to China, please contact our suggested distributor for assistance:
||Genetimes Technology, Inc.|
Unit 501, Building 18, No.481 Guiping Road, Xuhui District, Shanghai
Republic of China 200233
After contacting the Genetimes representative and determining handling fees, please
fax or email the completed two page DSHB order form to Genetimes for order processing.
Additionally, you may also contact Don Wang at Biopike for processing shipments to China:
After speaking with him and determining handling fees, you must fax Mr. Wang our completed 2
page order form so he can place the order.
Please contact the DSHB (email@example.com) with any questions.
Shipping to India
Due to customs issues, the DSHB cannot ship directly to India. To place an order, please contact Sundeep Amar at Biogenuix Medsystems Pvt. Ltd.
Biogenuix Medsystems Pvt. Ltd
Phone: +91-11-4875 4875
Fax: +91-11-2561 2008
412-B, 4th Floor
Jyoti Shikhar Towers
New Delhi 110058 INDIA
Terms and Conditions:
|Prices-DSHB ( for Non-profit Organizations)
||Prices in INR. Applicable sales tax extra
|Supernatant, 1.0 ml
|Concentrate(<1.0 ml), 0.1 ml
|Concentrate ( =1.0 ml), 1.0 ml
|Ascites (<1.0 ml) if available, 0.1 ml
|Ascites (=1.0 ml) if available, 1.0 ml
|Bioreactor Supernatant (<1.0 ml), 0.1 ml
|Bioreactor Supernatant (=1.0 ml), 1.0 ml
|FITC Tagged Supernatant, 40 µl
|Polyclonals, 10 µl
|Recombinant, 0.1 ml
1. The above rates are inclusive of shipping charge, custom duty and service charge of 17.5%.
2. The above rates have been calculated with a minimum delivery schedule of 4 weeks. Customers requesting urgent deliveries (subject to DSHB product availability) and customers from for-profit organizations should contact Biogenuix for a separate quote.
Shipping to Russia
To facilitate shipments to Russia,
please contact Vladimir Titov at Rusbiolink:
Shipping to Mexico
Due to customs issues, the DSHB cannot ship directly to Mexico. To place your DSHB order, please contact:
451 Tecate Rd. Suite 2G
Tecate, CA 91980
Shipping to Brazil
Due to customs clearance issues, we are unable to ship directly to Brazil. To place an order with the DSHB, please contact Luis Perin to arrange for delivery of your order.
Shipping to South Korea
Due to customs clearance issues, all orders to Korea must be routed through Biomax Co., Ltd. Please contact Jaehoon Kim to determine handling fees and to submit your order.
Shipping to Spain, Portugal and The Netherlands
The customs clearance process in Spain, Portugal and The Netherlands has changed, and FedEx can no longer provide broker services. Therefore all orders to these countries will be shipped via UPS. As a result, current shipment costs to Spain, Portugal and The Netherlands will be $35; with substantially higher rates for out of delivery service areas. These costs are subject to change. Please note: The DSHB cannot ship dry ice via UPS, so any hybridoma cell line purchase shipping to Spain, Portugal or The Netherlands must be for growing cells. Please contact us with any questions.
The Developmental Studies Hybridoma Bank accepts payment by VISA, MasterCard and American Express, by check in US dollars drawn on a US bank, by international money order or by wire transfer. To pay by credit card, please provide card details with your order including card number, expiration date, cardholder name, and the email address where the payment receipt should be sent. For other payment methods, please include a purchase order number from your institution with your order.
Regarding wire transfer payments, all bank fees are to be paid by your institution. When generating payment by wire, all transfers using SWIFT must be made with the “OUR” instruction code in order to make a full payment. We will carry forward any transaction fees deducted from payments received and include them on subsequent invoices.
For questions regarding the payment status of an order or account, payment options, copies of invoices, and general accounting information including W-9s and vendor-related issues, please contact:
||Developmental Studies Hybridoma Bank|
Department of Biology
The University of Iowa
028 Biology Building East
Iowa City, IA 52242
Phone: 319 335-1058
Fax: 319 335-2077
The DSHB warrants the efficacy of products only to the extent of the characterization provided in the data sheets by replacement or refund. The customer assumes all risks and responsibilities in connection with receipt, storage and use of materials from the DSHB. The DSHB warranty is for 30 days and shall not exceed purchase and handling costs.
Acknowledging the DSHB
We have been asked by NICHD to ensure that all investigators include an acknowledgment in publications that benefit from the use of the DSHB's products. We suggest that the following statement be used:
“The (select: hybridoma, monoclonal antibody, or protein capture reagent,) developed by [Investigator(s) or Institution] was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242.”
Please send copies of all publications resulting from the use of Bank products to:
||Developmental Studies Hybridoma Bank|
Department of Biology
The University of Iowa
028 Biology Building East
Iowa City, IA 52242