Toll-like receptors (TLRs) are part of the family of pattern recognition receptors (PRRs) that evolved to recognize pathogen-associated molecular patterns (PAMPs). A variety of PAMPs have been identified, from various microbial components to viral molecules. E.coli (Escherichia coli) has been widely used as a prototype Gram-negative bacteria with the capacity to stimulate TLRs, in particular to elicit TLR4-dependent immune responses. This receptor mainly recognizes an essential component of the outer membrane of Gram-negative bacteria: lipopolysaccharide (LPS). In vitro stimulation of immune cells for cytokine production can be achieved with the use of commercially-available purified TLR agonists, including LPS. Here we show how to prepare an alternative stimulator of TLR4 in a very fast an inexpensive way, paraformaldehyde-fixed preparations of E. coli. This reagent is a powerful tool for the in vitro activation of TLR4 that can be quantified and therefore used in defined amounts. Moreover, it is a safe reagent (the E.coli cells are fixed) and it is easy to prepare in large quantities and to store. Other microorganisms such as yeasts can also be fixed in this way.