If the antibodies are for double (or more) staining, it is possible to conjugate the antibodies with a fluorophore (Alexa 488 for instance) and by this there is no need of doing a secondary antibody.
For this, it is very important not to have any serum that contains also IgG. It is possible to do one of three things:
- Use DMEM without serum for the last two days: taking the cells out, centrifuging them and putting them in the serum free medium. The cells will not grow and suffer a bit.
- Another possibility is to use DCCM1 (there is also 2 and LPM) from “Biological industries”. This will allow you to also grow them a bit or step-by-step acclimate them to a serum free medium. http://www.bioind.com/Htmls/article.aspx?C2004=12426&BSP=12419&BSS51=12018
- Cleaning the sup with ProteinA, the FCS/FBS is low in IgG (200~500mg/L).
- 400 ml Iscoves DMEM
- 100 ml Fetal Bovine Serum (Heat Inactivated)
- 5 ml PenStrp
[For Pax7 add 1% Glu]
Filter all in the Biological hood.
Initiation of Culture:
- Thaw cells by rapid agitation in a 370 water bath. (Thawing should be rapid – within 40-60 sec).
- Transfer cell suspension into 0.5ml of fresh medium in a centrifuge tube.
- Centrifuge cells at 1200g for 7 min
- Draw off medium containing the freezing additive.
- Resuspend cells in approximately 1 ml of fresh media.
- Transfer to 50ml flask with 10 fresh media.
- Check for confluence and viability for the next few days.
- When cell density is 106 cells per ml (almost completely covering the bottom of the dish) split them 1:10 or 1:20 into 200ml. usually they need to be split every 3-4 days. Don’t let them grow too long without splitting or they will die!!
- Always keep a backup flask while growing so there will be no need to thaw new cells:
- Empty the flask from which you split from, put fresh media, these cells will take more time to grow (a week or so).
- Handle this culture in different days from which you handle the regular growing one, in order to avoid the contamination of both.
NOTE: these are non-adherent cells, which grow in suspension. In order to split them you just divide the medium into a new flask.
Collection of antibodies:
- To collect the antibody, let the cells grow until they get to 80-90% “confluence” (note that they don’t stick) and media turns yellow (takes about 3-4 days from split). Cells should be still alive and well. Centrifuge the cultures at 1000g for 10 min and save the supernatant.
- Freeze the supernatant in 1ml (or larger) aliquots.
- Try it at 1:10, 1:5, and 1:2, see what works best.
(There is separate protocol for concentration of the antibody)
Freezing away hybridomas:
Use cells that are healthy and rapidly dividing.
- Transfer cells into chilled centrifuge tube.
- Spin at 1200g for 7 min at 40C.
- Remove supernatant and resuspend the pellet in 8%DMSO/ 92%FBS (40C).
- Freeze in 0.5/1 ml aliquots (final cincetration of 5X105–107, from fully growing 10ml, split into 2).
Growing without Serum:
- Medium: DCCM with 2mM Glu + 1% PenSrtp.
- Spin cells, resuspend with DCCM and spin again.
- Resuspend with medium and grow with 200ml flasks.