Hello everyone,
I'm curently working on HaCaT cells which require low calcium medium to return to undifferentiate state. To do so, I need to chelate my FBS for 1h with chelex resin among other things.
Typical protocol suggests to chelate FBS, add to medium, then sterile filter the complete medium.
As only the FBS + chelex is not sterile and because filters are not that cheap, I rather filter sterilize a complete bottle of chelated FBS and then use it to complete my medium bottle.
Problem is the resin is clogging my 0.22µm filter, even 0.45µm get clogged before getting the 500mL of FBS passed through...
I was wondering if anyone had come across this problem and may have a handy solution.
- should I centrifuge the bottle before filtration and leave a dead volume ?
(i tried 5min 1500g but it didn't help much) maybe go higher ?
- maybe pass the FBS through a 80µm cellular sieve ?
- any ideas ?
Thanks for your time,
Philippe.
maybe prefilter the FBS+resin on a paper filter (3MM whatmann) by gravity then filter the FBS.
There are a few strategies you can try to improve the filtration process:
Pre-filtration or Centrifugation:
As you suggested, centrifugation can help remove larger particles before filtration. You might need to optimize the centrifugation conditions, such as increasing the speed or duration.
Pre-filtration through a larger pore size filter (e.g., 80µm) or a cell strainer can also help remove larger particles before using the 0.22µm filter.
Multiple Filtration Steps:
Consider using a stepwise filtration approach. For example, you can first filter through a larger pore size (e.g., 0.45µm) and then use the filtrate for a subsequent filtration through a 0.22µm filter. This can reduce the load on the finer filter.
Filter Washing:
If the filter becomes clogged, you might try washing it with an appropriate solvent to remove the accumulated material. This could be done during or after the filtration process.
Use of Filter Aids:
Some researchers use filter aids to enhance filtration. For example, diatomaceous earth or filter aid pads can sometimes be used in conjunction with the filter to improve flow.
Optimizing Chelex Resin Chelation:
Ensure that the chelex resin chelation process is optimized to minimize the presence of particles. You might want to try different incubation times or concentrations to find the most effective conditions.
- Badges
- Science topic
- Similar topics
- Chemistry
- Biochemistry
- Biochemical Techniques
- Blotting Techniques
- Immunoblotting
- Dyes