International Histocompatibility Working Group

Frequently Asked Questions

IHWG technician assessing the health of cells in culture

Irina Boguslavski, research technician, is visually assessing the health of the cells in culture. All flasks are labeled with barcoded cell line ID number and name, dates and tracing information, and visually inspected for turbidity, color change, over-proliferation, etc.

Photo by Emil Madraimov / IHWG

What method was used to type HLA genes of the IHWG cell lines?

The typing method utilized depended on which particular workshop the samples were collected under. 

During the 10th workshop (1987) Restriction Fragment Length Polymorphism (RFLP) typing technology was used.

During the 11th & 13th workshop (1991 & 2002)  PCR/Sequence Specific Oligonucleotide probe (SSOP) typing technology was used.

During the 12th & 13th workshop (1996 & 2002)  Sequence Based typing was utilized.

Select cell lines from the IHWG Cell and Gene Bank inventory are being retyped using the Next-Generation Sequencing (NGS) at the 17th workshop (2017).  

What language do I use to reference these cell lines in a manuscript?

Please indicate that the cell lines were previously collected and studied under International Histocompatibility Workshops & Conferences and obtained directly from the International Histocompatibility Working Group (IHWG) in Seattle, WA (www.ihwg.org).

What is the expiration date on IHWG cell lines and DNA?

Neither B-LCL or DNA have expiration dates.

Frozen cells are viable for at least 20 years from our experience, when kept in the appropriate conditions (LN2). When expanding the frozen cells, it is best to freeze down 5 million cell aliquots first, before you use the cell line for your research. Growing cells are viable for 2-3 months, after which they tend to have growth issues due to the number of passages.

Frozen DNA samples continue to have high integrity even after 15 years from our experience, when kept in the appropriate conditions (-20 or -80°C) and the samples are not put through too many freeze-thaw cycles. The more times a DNA sample goes through a freeze-thaw cycle, the more risk there to cause shearing and degradation. When receiving frozen DNA, it is best to thaw the sample and freeze down a number of aliquots first before use.

What QC assays are used to test IHWG cell lines and DNA?

Absence of Mycoplasma: The IHWG Cell and Gene bank utilizes the Invivogen PlasmoTest, a cell-based colorimetric assay, for detection of presence of mycoplasma. If positive results are detected using the Invivogen kit, the R&D Systems’ Mycoprobe Kit is employed as an accurate, sensitive RNA-based method for confirmation. With the Mycoprobe kit, the presence of Mycoplasma ribosomal 16S RNA is detected by a colorimetric signal amplification system.  

Absence of Bacteria and Fungus: Visual inspection of cultures is usually sufficient to detect whether gross bacterial or fungal contamination, evidenced by turbidity, alterations in cell line appearance and/or color changes in the medium, has occurred. Any suspicious cultures are tested in a reference laboratory for identification of the microorganism and aid in efforts to prevent and contain future infections. All suspicious or verified contaminated cultures are then discarded.

Cell Line Identity: Cell lines may be positively identified using a variety of methods, depending on the level of resolution required (family versus unrelated cell lines) and the number of markers necessary to uniquely identify a given cell line.

HLA typing represents an established method of identification, and many samples in the Cell Bank inventory have historical HLA typing data to serve as a QC benchmark. Various HLA typing techniques (sequencing-based typing [SBT], SSOP and SSP) may be used to assess alleles of the HLA-A, B, C, DR, DQ and DP loci.

Microsatellite (Msat) typing is used to detect stretches of DNA consisting of tandem repeats of a simple nucleotide sequence. These repeats can be easily amplified by PCR and characterized. The detection of multiple Msat loci provides a convenient DNA fingerprint of a given cell line. STRs are tetrameric repeat sequences that can be rapidly determined using a commercial kit. STR alleles are discrete, behave according to known principles of population genetics, and are digital and therefore ideally suited for computer databases.

ABO/sex typing utilizes a PCR reaction for identifying the blood type and sex of an individual sample. This information is used to confirm identity by referencing the sample history.

DNA Purity: Gel electrophoresis is used to identify the size and integrity of genomic DNA. Degraded DNA, consisting of many small fragments, will result in a “smeared” appearance on the gel following electrophoresis.

An OD 260/280 ratio is used to indicate DNA purity. For purified DNA, the 260/280 value should be 1.8, though a range of 1.6 and 2.0 is acceptable. RNA or protein contamination may cause OD 260/280 ratio shift to lower than 1.6 (normally indicating protein contamination) or more than 2.0 (normally indicating RNA contamination).

How should IHWG cell lines be cultured after shipment?

Your shipment will contain either frozen vials or growing cultures of B-lymphoblastoid cell lines (B-LCL). The cells were grown in our laboratory in RPMI complete medium (Gibco) supplemented with 15% fetal bovine serum (FBS; Gibco/Invitrogen Corp) and 1% 100 mM sodium pyruvate (Gibco). For international shipments, the sample has been frozen in 15% Pooled Human Serum (PHS; Valley Biomedical Corp) to avoid custom issues using bovine materials.

The cells have been quality control tested for mycoplasma, bacteria and other visible contaminants. We use the InvivoGen PlasmoTest kit for detection of mycoplasma. This method detects a metabolic by-product unique to mycoplasmas, but is only useful in detecting viable mycoplasma organisms. We recommend that the cultures be further screened upon receipt using a mycoplasm detection method of your choosing.

We recommend the following additional procedures upon receipt of the cell lines:

Note: Frozen vials should contain approximately 5 x 106 total cells; growing cultures have been shipped at 5 x 106 total cells but somewhat fewer may actually be recovered upon receipt.

  1. For growing cultures, proceed to Step 4. For frozen vials, thaw and suspend in 10ml culture medium. 
  2. Centrifuge the cells at 1200 RPM (250 g relative centrifugal force, RCF) for 7 minutes. 
  3. Decant supernatant and re-suspend the cell pellet in 10 ml culture medium. 
  4. Centrifuge the cells at 1200 RPM (250 g RCF) for 7 minutes. 
  5. Decant the supernatant and re-suspend the cell pellet in 5-10 ml culture medium. Transfer to a 25 cm2 vented (0.2 µ pore size) culture flask. We recommend supplementing the medium with 15% FBS (fetal bovine serum), as this is the concentration in which the LCL have been grown. 
  6. Incubate at 37º C in 5% CO2. Cell lines will recover and proliferate at different rates. We recommend daily monitoring until the cells have begun to show a healthy proliferation pattern.

Culture tips and guidelines:

Our cells are sensitive to antibiotics and should be grown with no antibiotics in the culture media.

The culture media should not have HEPES in it as this will slow cell growth.

Please be aware that different cell lines recover at different rates. Some take a day and some take more then a week to begin healthy growth.

Here are some different culture techniques you can try to help with any growth issues:

  • If the cultures are in T25 flasks, make sure there is no more then 25-30 mls of media. 
  • If there are growth issues after 1-2 weeks after thawing, to help encourage growth you can either remove most of the media and replace with 3-5mls of fresh media in the same flask, or move them to a T75 flask after removing most of the media and add 5mls of fresh media. 
  • If there are large clumps, but you are not seeing much growth, remove most of the media then break up the clumps with a pipette and then add 3-5mls of fresh media. 
  • If the culture has tiny, dusty looking cells 1 or more weeks after thawing, and the media is turning yellow frequently, remove most of the media and add 20mls of fresh media. Continue with this “changing out” until cells settle down and start clumping again. 
  • Laying the flask down instead of having it upright will also sometimes help with growth issues.

How should IHWG DNA be handled after shipment?

Check each label to verify that all samples are present. Please contact us immediately with any question regarding sample identity or problems encountered with the shipment.

“Quick-spin” each tube to bring down the DNA droplets. It is strongly suggested that the concentration of DNA in each sample be verified in your laboratory to ensure that no DNA has been lost during shipment. Aliquot each sample to “working” tubes containing the desired amount of DNA for testing. Freeze all tubes that will not be used immediately at -20ºC or lower. DNA to be used within 2 weeks may be stored at 4ºC. Avoid frequent freeze-thawing of individual DNA samples.
 

What are your terms and conditions?

For applicable terms and conditions of use of IHWG material, please select material and institution type below:


All Cell Lines:

All DNA: