The sample volume of cell culture supernatant is very limited. What is the minimum volume in microliters that can be used with the ELISA kit?

Topping up the dilution is a common approach, but it depends on whether your sample concentration is high enough. For example, if IFN-γ stimulation yields only a few dozen pg/mL, diluting it another 1.7-fold could bring it close to the lower limit of quantification. It is recommended to first test the undiluted supernatant from the same batch of cells alongside a 1:2 dilution to see whether the signals are parallel; if they are not parallel, you cannot simply top up the volume. If the sample volume is limited, it is best to switch to a high-sensitivity kit or a version with a nominal sample volume of 50 μL. Although it is more expensive, primary cell samples are more precious. You can also optimize the experiment by reducing the volume retained for testing, or pool technical replicates from the same treatment group, but biological replicates must not be pooled.

I have encountered similar issues. Using 60 μL for duplicate wells is quite tight. If the kit requires 100 μL, reducing it to 50 μL means the standard curve must also be run at 50 μL, and you must verify whether the standard curve OD remains within the linear range. Generally, manufacturers do not recommend changing the intra-well volume, as incubation diffusion and plate wash background will change. The safer approach is to contact technical support and ask whether there is a 50 μL sample version or a high-sensitivity protocol. Some imported brands allow dilution of culture supernatant 1:2 with diluent, but you need to record the dilution factor and multiply by the coefficient in the final result. 25 μL is even less recommended, as pipette tip error and intra-well liquid level differences have a major impact.

If the sample volume is insufficient and is made up with diluent, this is not "falsification." In essence, it is predilution, which is acceptable quantitatively, but attention must be paid to the fact that the matrix ratio has changed. For example, if you take 30 μL of sample + 70 μL of diluent, the sample is diluted 3.33-fold, and the proteins and interfering substances in the supernatant are also diluted along with it, while the standard curve still uses 100 μL of standard, resulting in a slight matrix difference. To confirm this, you can prepare a standard curve spiked with culture medium in the same proportion as the sample, rather than simple diluent. If time permits, switching to a 50 μL system or a high-sensitivity kit is more reliable.

It is recommended to reserve more supernatant in the experimental design. Primary cell stimulation experiments typically use 3 replicate wells per condition, and IFN-γ detection must also leave room for other indicators, so 60 μL will be very tight. Consider increasing the seeding density, reducing the culture medium volume, or adding stimulation wells and pooling the supernatant. Regarding the kit, prioritize screening those with a nominal sample volume of 50 μL or high-sensitivity kits, and ask the supplier for an electronic version of the instructions to confirm the minimum sample volume. Even if the kit specifies 100 μL, some technical support representatives may provide a low-volume protocol, but you will need to validate the parallelism of the standard curve yourself.

I would not recommend directly reducing the sample to 25 μL and running the assay without validation. In ELISA instructions, the sample volume is usually matched to the standard curve volume. Reducing the sample volume will cause the dilution ratio of the standard curve and the samples to be inconsistent, making quantification prone to being low or causing well-to-well jumping. Since your supernatant is only 60 μL, a more feasible approach is to bring the sample up to 100 μL with standard diluent, which is equivalent to a 1:1.67 dilution, and then multiply the readout by the correction factor; however, the premise is that the IFN-γ concentration in the sample must not be too low. It is best to run several preliminary tests first, and for a protocol using the same 50 μL volume, ask the manufacturer whether it can be modified.