. Author manuscript; available in PMC: 2020 Jun 1.

Published in final edited form as: Curr Protoc Microbiol. 2019 Feb 18;53(1):e78. doi: 10.1002/cpmc.78

Table 1:

Troubleshooting Guide



Problem	Possible cause	Possible solutions
Barrier development is minimal (TEER < 10 ohms* cm²)	Contamination	Check under a microscope for the presence of bacteria or fungi. Remember to include 1% penicillin-streptomycin in your media.
	Bad or expired culture media	Prepare fresh culture media.
	Wrong pore size inserts	For hCMEC/D3, TEER development is slow and minimal when using pore sizes of 3-micron or less. Use an 8-micron pore insert.
	hCMEC/D3 seeding density too low	Review your numbers for the initial hCMEC/D3 seeding density. If too low, cells won’t establish a monolayer.

TEER drops suddenly	Contamination	Check under a microscope for the presence of bacteria or fungi. Remember to include 1% penicillin-streptomycin in your media.
	During insert manipulation (media replacement, TEER measurement, etc.) the monolayer or membrane broke	Note which insert is the problem. If the monolayer was only disrupted, the TEER will rebound within hours, but if the membrane was broken, the TEER will remain low. If the latter discard that insert.
	Too high of an inoculum.	In 0.9 cm² inserts, more than 10⁷ C. neoformans cells will cause the barrier to break down.

CFUs arising from control wells	Cross-contamination from insert manipulation	When replacing media, make sure you use sterile tips. When measuring TEER, clean the electrodes with ethanol and PBS between readings. Make sure the PBS or other media you use is sterile.
	Using contaminated PBS for plating dilutions	Make sure the PBS (or another diluent) you are using is sterile.

No CFUs obtained from any wells	Inoculum too low	Increase the C. neoformans inoculum.
	Wrong agar plates	Make sure you are using YPD plates for CFU determination.
	Wrong size pore insert	Make sure you are using 8-micron (or larger) pore inserts.

Absolute transmigration varies widely between replicates	Different ratios of serum used during opsonization	Make sure you use the same ratio of cryptococcal cells to 40% serum between your replicates.
	OD₆₀₀ too different and/or age of cryptococcal starter culture too old	When using different strains within an experiment, or the same strain between experiment, make sure to grow them to OD₆₀₀ of 0.8 – 1.2 (log phase). Also, cells from old plates (> 4 weeks) behave heterogeneously, so make sure to start your culture from freshly streaked cells.