I'm performing FACS on e coli, but the cells are clumping together so each event is multiple cells.E
EDIT: I determined the problem. Bacteria must be gated on SSC threshold, NOT FSC threshold as is standard for eukaryotic cells.
I ran a control where I had one flask of e coli expressing GFP, and one flask expressing RFP. Run separately, I got a clean population of either high-GFP fluorescent events, or high RFP fluorescent events. If I mix 10uL of each population into 200uL TE buffer and immediately run that sample, all the events have high RFP AND high GFP fluorescence, indicating the cells are sticking together immediately upon mixing.
Cell line: ER2566 (NEB)
F- λ- fhuA2 [lon] ompT lacZ::T7 gene 1 gal sulA11 Δ(mcrC-mrr)114::IS10 R(mcr-73::miniTn10-TetS)2 R(zgb-210::Tn10)(TetS) endA1 [dcm]
Host strain for the expression of a target gene cloned in the pTYB vectors.
Carry a chromosomal copy of the T7 RNA polymerase gene inserted into lacZ gene and thus under the control of the lac promoter. In the absence of IPTG induction expression of T7 RNA polymerase is suppressed by the binding of lac I repressor to the lac promoter.
Deficient in both lon and ompT proteases.
Growth media: M9 minimal media
FACS running media: TE
I know there are anti-clumping agents for yeast and mammalian cells, but I haven't come across one for bacteria.
Can anyone recommend:
- an e coli protein expression strain that is known to not clump
- an anti-clumping agent for bacteria
- any other possible solutions
?
Thank you!
Some things to consider, May be you are growing your cells more than 16-18 H. Or change your liquid media, Muller-Hunton broth is known to induce more clumping than nutrient broth, so you can change your broth and incubate by shaking all the time.
For other bacteria, addition of Glycerol or tween 80 can inhibit clumping. Or for some experiement swhen you want to observe separated bacteris (Example by fluorescent mucroscopy) you can syringe your suspension 20 to 30 time by insulin syringe.
Good luck
Hello Amanda,
We regularly use mannose at 1 mM to block the clumping of yeast cells. SInce most fo the clumping occurs due to glycoproteins interacting maybe you coudl try adding mannose?
Best wishes,
Philippe Urban
It may seem rough, but when I was working with mycobacterium we used press liquid cultures through a needle to de-clump the bacteria before infection. Depending on the severity of the clumping this may work. But I agree with those above that growth conditions may be the cause.
Generally the pressure from the needle would be sufficient, but if additional de-clumping was needed we would turn to progressively smaller filters, starting at 50um and going down to 5um.
Best of luck
@Geno Exactly what we did by 20 to 30 time by seringing and passing the suspension through insulin syringe. It's efficious for intrecellular infection with fluorescent bacilli and give excellent results mainly when we want to colocalize the M Tb bacilli in some cellular compartments.
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