Protocols for Using Human Fibroblasts Expressing Human bFGF as Feeder Cells for iPSCs

New Product of the Week: Anti-GFP Polyclonal Antibody 100ug ABP-PAB-PAGFP10 $175.00.

Allele Biotech has introduced the highly efficient GFP-Trap for GFP fusion protein pull-down, and a monoclonal anti-GFP antibody for detecting GFP-fusion proteins after Immunoprecipitation with GFP-Trap. Just launched this week, the anti-GFP polyclonal antibodies provide an alternative method for analyzing the isolated proteins.

Pre-announcement: Allele Biotech will launch a FAQ and a User Forum online where you can also find common protocols in focus areas and exchange ideas with us or others.

1. Thaw one vial of irradiated feeder cells by swirling gently in 37oC water bath until all of the contents are thawed. One vial of 2×10^6 cells is sufficient to prepare two10-cm dishes, or two 6-well or 12-well plates (about 3-4×10^4/cm2).
2. Spray vial with 70% ethanol and wipe dry before placing in tissue culture hood.
3. Gently add 1 ml prewarmed feeder cell medium (alphaMEM or DMEM/F12 with 10% FBS), mix with contents of cryovial and transfer into 15-ml conical tube containing 4 ml prewarmed feeder cell medium.
4. Centrifuge the cells at 200g at room temperature for 5 min and discard the supernatant.
5. Resuspend the feeder cells in 12 ml feeder cell medium. If using a 6-well plate: add 1 ml of feeder cell suspension to each well of the 6-well plate containing 1 ml fresh feeder cell media per well. If using a 10-cm tissue culture dish: add 6 ml of feeder cell suspension to 10-cm tissue culture dish containing 6 ml fresh feeder cell media. If using a 12-well plate: add 0.5 ml feeder cell suspension to each well of 12-well plate containing 1 ml fresh feeder cell media per well. Gently shake the dish left/right and up/down 10-20 times without swirling the plate to evenly distribute the cells across the plate.
6. Incubate the cells in 37 1C, 5% CO2, overnight.
CRITICAL STEP When moving the feeder cell plates from the tissue culture hood to incubator, do not swirl the medium, as this tends to cause the cells to accumulate in the center. Immediately after placing the plates in the incubator, slide the plates forward and backward (2–3 cm) two times, then left to right (2–3 cm) two times to ensure equal distribution of the cells. Use within 5–7 days.
7. Split stem cells (~2.5 x 10^5 to 5 x 10^5 cells, or ~10% confluence) into plate with feeder cells: aspirate medium from ESC or iPSC, wash with PBS and add 0.5 ml of 0.05% trypsin. Incubate at 37oC, 5% CO2, for 5 min.
8. Inactivate trypsin with 3 ml stem cell medium (e.g. DMEM + 20% knockout serum replacement), and collect cell clumps in 15-ml conical tube avoiding making single cell suspension because ESC tends to die in single cell form.
9. Centrifuge at 200g at room temperature for 4 min.
10. Aspirate feeder medium from feeder plates (cells incubated in Step 6), rinse with one ml of stem cell medium and add 5 ml of stem cell medium and return to incubator.
11. Aspirate and discard supernatant from the conical tube in Step 8, resuspend cells in 5 ml stem cell medium, gently dispense the cell pellet three times, add to feeder cell wells or dishes.
12. Incubate stem cells grown on feeder cells at 37oC, 5% CO2, for 48 h.
13. Aspirate medium and replace with stem cell medium every day; if iPSC colony number is low, replace medium every two days.

Tags: bFGF, DMEM/F12, FAQ, feeder cells, forum, human fibroblasts, iPS cells, iPS protocol, iPSCs, New Product of the week, protocol, stem cells

Nobel Prize in Medicine Awarded to Discovery of Telomere and Telomerase

Elizabeth H. Blackburn, Carol W. Greider and Jack W. Szostak are credited with discovering how telomeres work and the function of telomerase. “As cells divide, chromosomes need to be replicated perfectly. Work by the researchers determined that telomeres protect DNA from degradation in the process, and that telomerase maintains the telomeres,” as reported by CNN.

Carol Greider was a student of Blackburn, and Szostak collaborated with the Blackburn group 20 years ago and has since left that field. Still remember going to Blackburn’s seminar as part of the molecular biology seminar series at USC in the early 90’s, and reading Szostak’s papers on aptamer selection while designing RNA aptamer selection schemes (SELEX) to find substrates of pre-mRNA splicing factors.

By JW

Product related note: Human telomerase gene TERT is provided on lentiviral vectors to increase efficiency of generating iPS cells.

Tags: aptamer, cancer research, chromosome, iPS cells, iPSC generation, lentiviral vector iPSCs, Medicine, Nobel Prize, Selex, telomerase, telomere

The economy recession is most likely over, says who?

The economy recession is most likely over, or so says the federal reserve chairman Ben Bernanke. Do you feel it? Are you seeing increased job opportunities when you leave your current lab or security if you have a post-postdoc position? In our industry, where the health of the economy is mostly measured by research budgets of individual labs or research groups, occasionally by budgets for contracting or licensing fees, the change, if any, is still hard-to-find. But hiring at academic institutes like UCSD seems to have picked up lately, probably due to addition grants from the Obama administration’s stimulus programs. At the same time, individual NIH R1 grants have been creeping up to easily around 1 million a year, program grants 3-5 millions. With more stimulus money kicking in to academic labs this fall, it is expected that the situation will further improve. Comments welcome.

Notes about recent jobs in Pharma/Biotech: since our last blog about massive Pfizer layoff of scientists in 02-09-09, a major layoff in the big pharma sector came from Merck, which announced on 06-11-09 that it would cut 16,000 jobs after completing its merger with Shering Plough. On 09-14-09, Eli Lilly reported job cots of 5,500 or roughly 14% of its work force. There are areas in the country where people report about the economy as “I went to 2 grocery stores and 3 discount department stores over one weekend, and you could do cannon shooting practice in there without hitting a person.” Again comments welcome here, if you believe in a turnaround, or it is all doom and gloom to you. Btw, the History channel has been cranking up the 2012 theories for a couple of months now, if you like the doom and gloom theories.

Note added in proof: As reported in Science yesterday, “a new analysis of the grantsmaking process at the National Institutes of Health (NIH) lifts the veil on how many grant proposals are funded even though they fall below a cutoff based on peer-review scores…at least 19% of NIH’s basic research portfolio is funded for reasons that go beyond quality.”

Francis Collins On the Job

Dr. Collins did a town hall meeting style announcement his first day as NIH Director on Aug 17th, 2009. He laid out his view for the NIH: more funding (good), encouraging young scientists (good, average age for first own funding for US biologist is 42, not good), and staying open in communication with society it is serving.

The NIH has $30.9 billion budget for 09 and 2010 thanks to the stimulus addition of $10 billion/year. However, it will feel dried up after two years if the budget plan remains as is. The Obama administration does not seem to want increase the basic research but instead focus more on health care management.

Collins is a well admired director and established scientist. However, it may be a little concerning that he might be too much into “big science” and organized efforts. I don’t know what they teach in graduate classes now but from what I was told 20 years ago curiosity-driven science is the best science and that was what got the US to the dominant leadership in biomedical fields.

Talking about nurturing young scientists, big programs and big labs controlling most grants by proposing big science seem trendy these days. The fight to become one of the big guys in a small, crowded field is a really daunting path for young researchers to tread. The big guys have the say from publication to funding and often times the unpleasant thought and bitter taste of competing against a scientific juggernaut turn young researchers away.

Tags: big research, Dr. Francis Collins, NIH budget, NIH director, Obama's research policy, young researchers in US

Allele’s Online Community

Allele Biotech’s participation in social networking sites (Twitter, Facebook, and MySpace has been successful over the past few months! We initiated contact with our customers via these websites not only to provide an easy way to let people know about our newest products and promotions but, most importantly, to ensure better customer service through an easily accessible forum for questions, comments, and yes, even criticisms. We are not like those other companies that are so large that your business and opinions do not matter to us. We at Allele Biotech need and appreciate our customers and reward your patronage and participation in these social networking forums with special promotions and customer service that really lets you know we value your online community membership. Your research goals are our research goals!

Several times a week our tweets will inform you about great deals like FREE SHIPPING on select products ordered within a specific time frame.

Our regular blogs found on all three of our sites are used to converse on a variety of topics from SBIR grants to fluorescent proteins to skin care!

On facebook and myspace you can submit technical questions on protocol or products and receive SAME DAY answers; you may also send comments and suggestions for improvement which will be seen by our head scientist and executives. Your opinion counts at Allele! We began this networking concept as not only a way to better reach our customers but, more importantly, as a way for them to better reach us.

Become a friend, fan, or follower to any one of the sites today and receive a $30 discount off your next order!

Tags: Allele customer service, allele social marketing, allele tweets, Facebook, myspace, research goals, same day troubleshooting, twitter