Hi Nils, w

hat is your method of freezing/thawing?

Maybe this could be helpful

Article Guidelines for the use of flow cytometry and cell sorting in...

"However, even if precautions are taken, freezing cells inevitably influences cellular biology and key parameters that are likely to be a key element of the study such as viability, immunological capacity and responsiveness, and ability to be expanded in vitro. The properties of thawed cells might be significantly different to their freshly isolated counterparts. These potential issues and limitations therefore need to be taken into consideration. Whenever possible, one should determine the effect of freezing and thawing on the key biological and immunological readouts before embarking on an experimental program that stores frozen samples.

Maintaining the highest level of viability requires cells to be frozen in a cryoprotective solution. DMSO is a commonly used solution that, when used at a concentration between 5 and 10% v/v in an appropriate medium, retains a high level of viability after storage. One technical point to consider is that the best recovery is achieved with a gradual freezing process, i.e., lowering the temperature of the cells by 1 to 2°C/min. This procedure is intentionally slow in order to prevent the formation of ice crystals and cell rupture. Higher concentrations of DMSO (up to 10% v/v) allows faster freezing and has been shown to deliver 85% post-thaw viability, with some variability between different types of leukemia. Automatic freezing techniques using temperature-controlled setups have been developed for the routine cytometry laboratory. In these systems, the cell samples are slowly moved down a tank of liquid nitrogen by a motor-driven spindle. Commercially available cell freezers are the most suitable appliances for this process. However, manual methods have been widely reported to give adequate results.

The thawing process is as important as the freezing one and must be done very rapidly, with active thawing being preferential to a passive one. Active thawing and other steps in the thawing process have been evaluated for leukocytes by Hønge et al. [69]. It should be appreciated that different cell types respond differently to thawing, and this needs to be taken into consideration during experimental design. As an example, Alsayed et al. [70] reported that myeloid leukemia cells recovered better than lymphoid leukemia cells. Immunophenotyping is an important and frequently used method in risk assessment and posttherapy follow-up in the clinical laboratory that requires a high degree of standardization and post-thaw viability tests in order to ensure that results are accurate and robust."

The loss of monocytes could be hypothesized because of their phagocytic activity of LPS. This hypothesis can be supported by two-three intermediate counts before the 18-hour one to highlight the progressiveness of the reduction. A stained smear with MGG might be helpful.

Also, if your research protocol allows it, try reducing the amount of LPS