The effect of whole blood logistics on neutrophil non-specific activation and kinetics ex vivo

Abstract While the exquisite sensitivity of neutrophils enables their rapid response to infection in vivo; this same sensitivity complicates the ex vivo study of neutrophils. Handling of neutrophils ex vivo is fraught with unwanted heterogeneity and alterations that can diminish the reproducibility of assays and limit what biological conclusions can be drawn. There is a need to better understand the influence of ex vivo procedures on neutrophil behavior to guide improved protocols for ex vivo neutrophil assessment to improve inter/intra-experimental variability. Here, we investigate how whole blood logistics (i.e., the procedure taken from whole blood collection to delivery of the samples to analytical labs and storage before neutrophil interrogation) affects neutrophil non-specific activation (i.e., baseline apoptosis and NETosis) and kinetics (i.e., activation over time). All the experiments (60+ whole blood neutrophil isolations across 36 blood donors) are performed by a single operator with optimized isolation and culture conditions, and automated image analysis, which together increase rigor and consistency. Our results reveal: i) Short-term storage (<8 h) of whole blood does not significantly affect neutrophil kinetics in subsequent two-dimensional (2D) cell culture; ii) Neutrophils from long-term storage (>24 h) in whole blood show significantly higher stability (i.e., less non-specific activation) compared to the control group with the isolated cells in 2D culture. iii) Neutrophils have greater non-specific activation and accelerated kinetic profiles when stored in whole blood beyond 48 h.


Introduction
Neutrophils, the most abundant immune cells in our body, form the rst line of defense in human immunity. While recently increased efforts have shifted towards studying the multifaceted role of neutrophils in many diseases, e.g., cancer and infections 1,2 , there is still a call for further understanding the basics of neutrophil kinetics including survival (or cell death), activation, and migration both in vivo and ex vivo 3 . For example, the commonly cited "short lifespan of neutrophils'' thought to be 7-9 h is actually the disappearance rate of neutrophils in the blood 4 , which does not necessarily account for the lifespan of neutrophils in different compartments (e.g., bone marrow, tissue) 3 . Recent studies indicate a long lifespan of neutrophils for up to 3-5 days in vivo 5 . When neutrophils are exposed to external insults (e.g., tissue injury) and/or invading substances, the innate immune cells can become activated and intervene through phagocytosis (i.e., ingestion of pathogens), secretion of antimicrobials via degranulation, and a newer third function -NETosis [formation and release of neutrophil extracellular traps (NETs)] 6 . The many potential triggers, the activation pathways, and the emerging functions of NETosis (e.g., suicidal NETosis versus vital NETosis) are still under investigation 7,8 .
Human immunity acquired critical plasticity and adaptability from exposure to constantly changing local challenges and conditions. Recent studies have shown that the environment plays a greater role in shaping the immune system compared to most heritable factors 9,10 . These insights underscore the importance of better understanding the interplay between environmental conditions and immune response. Furthermore, other groups have shown that whole blood collection method and anticoagulants, the storage temperature of neutrophils after isolation 11 , autologous signaling molecules 12 , oxygen tension in the culture environment 13,14 , and co-culture conditions 15 are all important factors to consider when assaying neutrophil behavior ex vivo (see summary in Table 1). Baseline neutrophil characteristics, including activation state, kinetic pro les, and functional outputs [e.g., reactive oxygen species (ROS) production, cytokine secretion] are in uenced by a multitude of tunable external factors and can quickly shift toward non-physiological conditions. Overall, these ndings suggest that neutrophils are sensitive to their culture environment, and optimizing these parameters can be helpful to make studies more rigorous and reproducible. However, these studies were limited by inconsistent isolation methods, variable culture conditions (e.g., media composition, culture times, cell stains), and multiple operators which can all lead to confounding variables that make downstream analysis inconsistent and parallel comparison of the conclusions (from different labs) di cult. During the in vivo to ex vivo transition neutrophils are subjected to a multitude of altered environmental conditions; therefore, baseline neutrophil kinetics are already affected and potentially skewed before and during interrogation in an assay, so identifying optimized cell culture and storage parameters is essential for generating robust and reliable data. These variations are further ampli ed when working with whole blood samples collected in the clinical setting when the time from blood collection to neutrophil interrogation (i.e., whole blood logistics) can range from hours to days depending on the nal entity/institution assaying for molecular and functional readouts. Establishing a standard set of practices for whole blood handling and culture/interrogation of neutrophils after blood collection can make results more comparable across experimental replicates, between different labs, and minimize unwanted heterogeneity.  15 Co-culture with blood endothelial cells increased neutrophil lifespan (via GM-CSF) and migration (via IL-6) in the in ammatory environment.
*Different isolation methods and culture conditions (including incubator, culture media, oxygen level, 2D vs 3D, culture time, and cell stains) are adopted from lab to lab, which makes the results di cult to be compared in parallel and to re ect the complete kinetic pro les of neutrophils (i.e., the latest understanding of neutrophil lifespan in vivo is 3-5 days).
Here, we seek to reduce non-speci c neutrophil activation (i.e., the baseline apoptosis and NETosis) and maintain their standard kinetic pro les (i.e., how the cells get activated over time in a speci c ex vivo environment) at baseline (i.e., immediately after isolation, prior to interrogation) while trying to understand the external parameters that in uence these characteristics. To this end, we identi ed and applied optimal neutrophil isolation strategies and 2D culture environment conditions (e.g., oil overlay, incubator system, length of culture, cell staining concentrations, and seeding densities) while investigating more downstream variables in neutrophil interrogation. We also investigate how disparities in the time between whole blood collection and neutrophil isolation prior to interrogation can contribute to baseline properties (e.g., non-speci c activation and kinetics) of isolated neutrophils. Additionally, to the best of our knowledge, no previous work has studied how long-term (up to 72 h) neutrophil storage in whole blood can in uence neutrophil behavior. This unique parameter is important to further elucidate since neutrophils are not always immediately isolated due to technical or logistical limitations (e.g., clinical settings or distant site of end point analysis). Through this work we highlight how neutrophil storage in whole blood at physiologic temperature (37 o C) across multiple days can minimize nonspeci c activation observed in time-matched 2D culture, while exhibiting accelerated kinetic pro les relative to baseline. All the wet lab work was completed by a single operator to minimize potential complications of operator-to-operator heterogeneity to increase the robustness of this study. Additionally, measurements were performed by an automated analysis pipeline to further reduce bias and increase the throughput of this work. This extensive study across 36 unique donors with 60+ whole blood neutrophil isolations from a single operator helps to clarify several optimal parameters for ex vivo whole blood handling and establishes a framework for exploring other modi able external factors that can affect neutrophil behavior.

Results
Neutrophil stages and the identi cation method Mature, healthy (non-activated) neutrophils take a unique multilobed-shaped nucleus, which gives the cells high malleability and motility 16 . When exposed to tissue injury or infection in vivo, neutrophils are activated, responding to the lesion in different types of cell death ( Fig. 1) -primarily apoptosis and NETosis 7,17 . Based on the mechanism of cell death, the shape of the neutrophil nuclei is distinguishably different. During apoptosis the chromatin condenses while during NETosis the chromatin decondenses 18 .
Apoptosis and NETosis can be distinguished by analyzing the shape of nuclei with a membranepermeable DNA stain (Hoechst) 19 . NETosis is known to have two distinct subtypes -suicidal NETosis (causing cell death) and vital NETosis (no immediate cell death) 7 . Suicidal NETosis features ruptured cytoplasmic membrane and release of diffused NETs. By contrast, vital NETosis features intact cytoplasmic membrane and release of stringy NETs. Release of NETs can be visualized by membraneimpermeable DNA stains (e.g., Sytox or propidium iodide) 19 . The rupture of cytoplasma membrane (in apoptosis and suicidal NETosis) can be visualized by phosphatidylserine stain (Annexin V) 18 .
In this work, we adopted Hoechst-Sytox-Annexin V (H-S-A) as a three-dye method. As summarized in Table 1, i) Hoechst is a DNA stain that penetrates intact plasma/nuclear membranes in presence of trace amounts of dimethyl sulfoxide (DMSO). Hoechst stains DNA of neutrophils during all stages, including healthy cells, early apoptosis, sec. necrosis, suicidal NETosis, and vital NETosis. It's worth noting that Hoechst shows little staining on NETs (decondensed chromatin) in the presence of Sytox, likely due to the competition between the two dyes and the dissociated structure of the chromatin. Hoescht staining is normal on condensed nuclei in sec. necrosis when co-stained with Sytox. Hoechst intercalates into the double strands of DNA in living cells, which affects many aspects of cell function. To this end, Hoechst was only used for end-point staining ( Supplementary Fig. 1). ii) Sytox Green is a DNA stain that is impermeable to intact plasma/nuclear membranes. When the plasma/nuclear membrane is compromised/ruptured, Sytox stains any DNA content exposed to the dye. Sytox stains the condensed chromatin of neutrophils in sec. necrosis, and NETs from neutrophils that undergo NETosis. iii) Annexin V stains phosphatidylserine (or the "Eat Me" molecules) on the plasma membrane of dying or dead cells and thus it stains neutrophils in early apoptosis, sec. necrosis, and suicidal NETosis. Sytox and Annexin V were used for real-time staining (i.e., the dye was added to the cell stock before cell seeding and present through a culture) to monitor neutrophil kinetics in culture ( Supplementary Fig. 1).
Next, we optimized the H-S-A staining protocol to minimize the potential effects that uorescence labeling has on normal cell function 21 . Here, we tested three different concentrations of the Sytox dye and the in uence of Sytox and Annexin V on each other ( Supplementary Fig. 4). For Sytox, 10000× and 5000× dilution made no signi cant difference on non-speci c activation and neutrophil kinetics compared to the no-dye control ( Supplementary Fig. 5). Sytox at 1000× dilution caused higher activation especially after 6 h from the initiation of cell culture ( Supplementary Fig. 4a, b). The combined use of Annexin V (20× dilution) with Sytox (5000× dilution) led to an increased Sytox signal but interestingly, there was no difference in the Annexin V signal compared to the Annexin V only control group ( Supplementary Fig. 4c, d). To minimize the artifact from real-time staining ( Supplementary Fig. 1a, b), we chose to use 10000× Sytox with 40× Annexin V. Hoechst was only used for end-point staining to avoid any artifacts in cell function and prevent DNA damage 22 .

Isolation method
Different isolation methods have been developed and used for isolation of primary neutrophils from whole blood (e.g., gradient neutrophil isolation with RBC sedimentation or lysis, negative selection-based neutrophil isolation with or without RBC lysis). Previous work has shown that different isolation methods can affect neutrophil phenotype and activity in functional assays; therefore, we explored two commonly used methods to identify a method best suited for this study 23 . Here, we compared two negative selection kits [MACSxpress (with RBC lysis) versus EasySep (without RBC lysis) following the manufacturer's instructions] ( Supplementary Fig. 6a). The results showed that the non-speci c activation right after isolation was comparable between the two kits but noticeably MACSxpress based isolation led to accelerated kinetics in 2D culture of the neutrophils ( Supplementary Fig. 6b, c). Therefore, we chose to use EasySep negative selection without RBC lysis in this work.
House-keeping factors in 2D culture of neutrophils Different research groups may use a range of culture conditions in their studies, which likely introduces variability and impedes reproducibility/comparability of the results between studies. Here we investigate how the selection of culture conditions may in uence non-speci c activation and neutrophil kinetics in standard 2D monoculture ex vivo. Based on our previous experience with microscale cultures, we recognized the importance that media evaporation, incubator system, and seeding density may all play key roles in maintaining proper cell behavior.
Microscale cell culture methods possess many advantages over bulk-scale (e.g., petri dish, culture ask) methods due to their ability to better recapitulate the parameters of in vivo cellular microenvironments (e.g., structural hierarchy, uidic dynamics, and mass transport). Due to the small volume (< 100 µl), media loss via evaporation becomes a signi cant variable that exerts stress to the cells in culture. We tested the isolated neutrophils in standard 2D monoculture from nine donors with and without oil overlay ( Supplementary Fig. 7a). In ve of the nine randomly selected donors, oil (especially silicone oil) overlay led to less non-speci c activation compared to the no-oil culture, as measured by the area fraction of the Sytox signal at the end of the stationary phase ( Supplementary Fig. 7b, see the de nition in Fig. 3a). For the remaining four donors, silicone oil overlay did not signi cantly affect the activation status of the neutrophils. There was no obvious pattern in which donor neutrophils would bene t from the silicone oil overlay. Therefore, for subsequent experiments we used a silicone oil (coded in tan color in the gures) overlay in 2D culture to get improved culture environment stability and consistency.
Next, we compared two incubator systems -a standard incubator (SI) versus an onstage incubator (OI) (Supplementary Fig. 8a). They all allow the operators to select and control the O 2 level, CO 2 level, temperature, and RH. Compared to a standard incubator, an onstage incubator allows optical access to the culture system on a microscope to enable real-time imaging. We interrogated two randomly selected donor neutrophils to study neutrophil activation and kinetics between these incubator conditions. Culture in the onstage incubator led to dramatically accelerated kinetics ( Supplementary Fig. 8b) compared to the standard incubator. The incubation phase (see the de nition in Fig. 3a) was dramatically shorter in the onstage incubator for both donors (~ 5 h) and reached the stationary phase (~ 25 h) much quicker than their standard incubator counterparts (~ 50 h) ( Supplementary Fig. 8b). Therefore, we chose to use a standard incubator in this work. For experimental work ows, the culture plate was stored in a standard incubator and was only taken out for imaging in the onstage incubator.
Finally, we investigated the possible in uence of cell seeding density on neutrophil kinetics. In a standard 384-well plate, 60000 (or 60K) cells per well achieves a near-con uent monolayer of neutrophils ( Supplementary Fig. 9a). Neutrophil seeding densities across a broad range (10K, 30K, 60K, and 200K per well) were trialed. When seeded at 10K or 30K cells per well, neutrophils did not show any difference in normalized activation ( Supplementary Fig. 9b). The highest seeding density (200K cells per well) showed higher total activation (~ 2-fold higher) compared to the 60K cells per well condition but lower activation (~ 60%) after normalization to per 10K cells ( Supplementary Fig. 9c). Overall, the near and less con uent seeding densities showed no signi cant in uence on non-speci c activation of neutrophils and the seeding density of 60K cells per well allowed more even and consistent distribution of the cells in a well from cell seeding and thus enabled easier/more reliable data collection and analysis.

Statistics of whole blood handling time
To study human neutrophils ex vivo, the typical work ow includes blood logistics (i.e., collection and transport), storage, processing, and interrogation (e.g., cell culture and assessment) (Fig. 2a). Each additional step before interrogation in the work ow introduces possible bottlenecks and delays that can contribute to the overall quality of the isolated cells used in the terminal assay. We evaluated the components of whole blood handling to identify key parameters that in uence baseline neutrophil apoptosis and NETosis, de ned as "non-speci c activation" after transfer to standard 2D cell culture. In the 2D culture, neutrophils were cultured in a standard incubator with and without oil overlays as described above (Fig. 2a). Blood was obtained from multiple sources including draws completed in our laboratory and through a collaborator's lab at the UW hospital (under an IRB approved protocol, see Methods). Across 36 unique blood donors, our results highlight a highly variable whole blood logistic time (t l ) and whole blood processing time (t p ) from case to case (Fig. 2b). t l ranged from 0.5 to 5 h depending on the e ciency of donor ID de-identi cation, the distance/communication between the blood collection locus and the lab performing the subsequent experiments (Fig. 2b). Similarly, t p ranged from 1 to 4.5 h depending on how many whole blood samples processed in an experiment and how many conditions in the following interrogation (Fig. 2b). On average, it took 4-5 h from blood collection to the start of neutrophil interrogation if no whole blood storage time (t s ) was added to the "dead time" (i.e., t l + t s + t p ) in this work performed by a single operator.

Short-time (< 8 h) whole blood storage
Clinical studies often require processing of blood samples but can be delayed due to the logistics of transport to third-party entities, so there is a need to better understand if whole blood storage time and conditions can affect the baseline characteristics of isolated cells. Recent work identi ed that whole blood storage time of samples drawn from pigs led to differences in ROS production, the level of NET formation, altered neutrophil survival kinetics, and modi ed antimicrobial activity 29 . Here, we look into how whole blood storage time, temperature, and method (i.e., stationary storage versus rotary storage) affect apoptosis and NETosis from samples collected from healthy human donors and patients with cancer. The analysis was broken into two distinct time periods, < 8 h (i.e., short-time storage) and > 24 h (i.e., long-time storage). Early experiments indicated that the 8 h mark constituted an in ection point at which neutrophil behavior could begin to change and coincided with a traditional 8 h work shift.
Considering the varying dead time of whole blood handling, it is important to understand rst how shorttime whole blood storage (with t s <8 h) may in uence non-speci c activation and neutrophil kinetics in standard 2D monoculture ex vivo (Fig. 3). From a whole blood sample with t l <1 h, we partitioned the blood into three groups: immediate isolation (i.e., t s = 0 h) (Fig. 3a), stationary storage (at RT for 2.5 h) ( Fig. 3b), and rotary storage (on a tube rotator at RT for 6.5 h) (Fig. 3c). Comparison between the stationary storage and rotary storage is limited by the 4 h time difference in storage time. This difference is attributed to practical limitations of a single operator carrying out all the tasks and material limitations (e.g., magnetic isolation kits) available to the operator. We further analyzed the kinetic pro le of the isolated neutrophils after culture in the unique culture conditions. In standard 2D monoculture, the isolated neutrophils showed three distinct phases in kinetics, which include i) incubation phase (low activation, 0-12 h), ii) exponential phase (fast activation, 12-42 h), and iii) stationary phase (saturated activation, > 42 h) (Fig. 3a). Our results indicate short whole blood storage times (< 8 h) did not increase non-speci c activation or signi cantly alter kinetics in 2D culture (Fig. 3).

Long-time (> 24 h) whole blood storage
Conversely, long-time whole blood storage times (t s >24 h) may in uence non-speci c activation of neutrophils (Fig. 4). Neutrophils stored at 37 o C, in a standard 2D monoculture environment, for greater than 24 h and up to 48 h showed increased levels of Sytox staining (Fig. 4a, b), which accounts for NETosis and secondary (sec.) necrosis (Fig. 1). In contrast, neutrophils stored in whole blood at 37 o C maintain relatively low levels of Sytox staining immediately after isolation, indicating low levels of nonspeci c activation (Fig. 4a, b). In all conditions, Annexin V staining, which measures apoptosis, sec. necrosis, and suicidal NETosis (Fig. 1), was not appreciably different. As cells undergo apoptosis and nuclear condensation occurs, the circularity of the multilobed neutrophil nucleus increases towards 1.00 (which indicates a round nucleus) ( Supplementary Fig. 2). The automated nuclear circularity analysis pipeline (Methods) developed here quanti ed the differences in circularity between the different storage conditions at the speci ed time points. Neutrophils that were immediately isolated and kept in culture at RT had increased circularity (i.e., more apoptosis) over the subsequent 48 h of culture (Fig. 4c, d).
Neutrophils stored in whole blood at 37 o C and subsequently isolated at the 24 h and 48 h time points had signi cantly less circularity (i.e., more healthy neutrophils with multilobed nucleus) when compared to their RT counterparts (Fig. 4c, d).
Next, we investigated the kinetic pro les of neutrophils in the different storage conditions and length of storage. For these studies, each whole blood sample, from three random donors (Prostate383, Prostate579, Healthy103), was partitioned into four groups -immediate isolation, and whole blood storage for 24 h, 48 h, and 72 h. Neutrophils in the immediate isolation condition were placed in 2D culture and incubated in RPMI + 10% FBS + 1% P/S (Fig. 4f Prostate383-i, Prostate579-i, Healthy103-i).
These neutrophils were monitored for the subsequent 72 h to assess their kinetic pro le. In two of the donors (Prostate383, Prostate579), the neutrophils stored in whole blood for 24 h were subsequently isolated and placed into 2D culture as described above for 72 h (Fig. 4f Prostate383-ii, Prostate579-ii) to assess their kinetic pro les. For the third donor (Healthy103), neutrophils stored in whole blood for 24 h, 48 h, and 72 h were subsequently isolated and placed into 2D culture as described above (Fig. 4f Healthy103-ii, Healthy103-iii, Healthy103-iv). For each donor, the circularity of nuclei of the neutrophils was measured to assess apoptosis (Fig. 4g) across the different time points and storage conditions. Neutrophils stored in whole blood for 24 h prior to isolation showed accelerated kinetics (Fig. 4f Prostate383-ii, Prostate579-ii, Healthy103-ii) compared to the immediate isolation control (Fig. 4f Prostate383-i, Prostate579-i, Healthy103-i) as their 0 h time point showed signi cantly higher levels of non-speci c activation (~ 10% vs 0%). Furthermore, neutrophils from the third donor (Healthy103) that were placed into culture at 48 h and 72 h post-whole blood storage showed even greater levels of kinetic acceleration, since they reached the stationary phase of their activation pro le early on in 2D culture ( Fig. 4f Healthy103-iii, Healthy103-iv). However, neutrophils stored in whole blood for 24 h had lower levels of apoptosis, as measured by nuclear circularity, compared to the 24 h time point for the immediate isolation condition for all three donors (Fig. 4g). For the third donor, the neutrophils stored in whole blood for 24 h, 48 h, and 72 h all show less apoptosis compared to the 24 h time point of the immediate isolation condition (Fig. 4g). For the third donor, we compared the level of activation of the neutrophils in each storage condition with respect to total time after isolation instead of time in 2D culture. Neutrophils in either 2D culture with RPMI + 10% FBS + 1% P/S or stored in whole blood showed similar levels of nonspeci c activation and apoptosis up till 24 h after isolation, but there were signi cantly lower levels of non-speci c activation and apoptosis in the whole blood storage condition between 24 to 48 h time points (Fig. 4h). By 72 h, both storage conditions showed high levels of neutrophil non-speci c activation and similar levels of apoptosis (Fig. 4h).

Whole blood storage temperature
Finally, we investigated the in uence of whole blood storage temperature on non-speci c activation and neutrophil kinetics. Across two donors, whole blood was partitioned into immediate isolation, and either  (Fig. 5a, b). Annexin staining between the immediate isolation condition and both storage conditions was not consistent between the two donors. For both donors, nuclei circularity analysis showed that there was signi cantly more apoptosis in the 37 o C storage condition compared to the RT storage condition (Fig. 5c).

Discussion
Neutrophils are ubiquitous in circulation and their unique roles in cellular immunity continue to be elucidated. In recent years, their role in a variety of disease states has become more apparent which coincides with increased usage of neutrophils ex vivo for interrogation in a breadth of functional assays and molecular characterizations. Delays in neutrophil interrogation from time of whole blood collection to nal assay processing are abundant due to inherent logistical hurdles, and potentially elongating factors associated with transport to end-user facilities. Heterogeneity in neutrophil biology can be attributed to a host of factors including the underlying disease state, intrinsic patient-to-patient differences, and as shown here -modi able external factors after whole blood collection and prior to nal interrogation in an assay of interest. Comprehensive characterization of neutrophils ex vivo has also been limited by poor ex vivo stability leading to non-speci c activation. High variability in the baseline non-speci c activation of neutrophils introduces variability that can make technical replicates and experimental replicates challenging to interpret or compare. Here we sought to elucidate how some of these factors -speci cally, time from collection prior to isolation, storage orientation, time, and temperature in uence baseline characteristics of isolated neutrophils in standard 2D cell culture. The wet-lab work for this study was completed by a single operator to reduce potential person-to-person variability in whole blood handling and cell culture.
Using an optimized trio of dyes (Hoechst, Sytox, Annexin V), we characterized how the above modi able factors contribute to the non-speci c activation state of isolated neutrophils by measuring apoptosis and NETosis through an automated image analysis pipeline across subsets of 36 unique blood donors. We identi ed that the EasySep negative selection method without RBC lysis yielded abundant neutrophils with minimal non-speci c activation and normal kinetic pro les. Based on our experience with microscale cultures in micro uidic devices we were cognizant of the in uence of evaporation, culture environment, and seeding density on cell behavior 20,[24][25][26] . We observed that minimizing evaporation with a layer of silicone oil on top of normal cell culture media decreased non-speci c activation of neutrophils from some donors. Furthermore, we observed that 2D culture in the standard CO 2 incubator showed more consistent and minimal neutrophil activation compared to an on-stage microscope incubator. Traditional incubators are better sealed and are less prone to shifts in O 2 levels, CO 2 levels, temperature, and RH in contrast to on-stage microscope incubators.
Through processing samples from healthy donors and patients with cancer across two collection sites, we observed that the average dead time (i.e., whole blood logistic time + storage time + processing time) from a single operator was about 5 h. Despite both collection sites being located within the same building, the high variability in dead time highlighted the need to better understand how even more disparate conditions (e.g., cross campus or cross institute delivery) could affect neutrophil behavior after storage in whole blood. Maintaining the whole blood in a rotary con guration was hypothesized to impose shear stress on the cells to better mimic the in vivo physiology of vasculature and hopefully improve neutrophil stability; however, whole blood storage (stationary or rotary) within 8 h (at RT) did not cause appreciable differences in non-speci c activation and neutrophil kinetics in standard 2D monoculture.
For storage beyond 24 h, neutrophils stored in whole blood (at 37 o C) showed signi cantly higher stability (i.e., less non-speci c activation) compared to the 2D culture controls. It is worth noting that we observed that long-time whole blood storage leads to decreased neutrophil yield from negative selection-based isolation methods. Additionally, neutrophils stored at 37 o C had less NETosis but had increased apoptosis compared to RT. While neutrophils can maintain low non-speci c activation in whole blood up to 48 h, a storage time > 48 h in whole blood leads to non-speci c activation at a level comparable to the cells in standard 2D monoculture for the same length of time. Neutrophils stored in whole blood for between 24-48 h also exhibited accelerated kinetics when placed in 2D culture compared to the immediate isolation control. Assessing neutrophil storage across multiple days highlights the importance of evaluating both the activation status and kinetics when trying to assess the baseline status of neutrophils. Despite showing minimal activation across all the long-time storage conditions (up to 72 h), which would suggest that any of these cells would be amenable to use in an ex vivo assay, the acceleration of the kinetic pro les of the neutrophils stored in whole blood > 24 h would introduce signi cant heterogeneity into a study and make potential functional readouts less comparable between experimental and biological replicates.

Study limitations
A limitation of this study is that only apoptosis and NETosis are studied while other functional readouts like ROS production or antimicrobial activity and molecular readouts are not analyzed. These differences would also provide insight into how whole blood logistics and short storage time conditions could affect these readouts; however, the observed fundamental differences in the baseline properties of the isolated neutrophils like the non-speci c activation status and kinetics over time we identi ed highlight the need to prioritize and account for these factors before more expensive and detailed characterization is completed.
All together our data illustrates the potential effects that externally modi able factors can have on neutrophil non-speci c activation and kinetics. Whole blood logistics are highly variable, and this variability will be unique to each institution and a liated third-party users who perform the end readouts. Our data suggest that minimizing the time from collection to assay to less than 8 h can help to improve reproducibility of readouts, since we observed no signi cant differences in non-speci c activation or kinetics of the neutrophils at these timepoints. Additionally, whole blood storage at 37 o C for up to 48 h can maintain the stability of neutrophils with minimal activation but with measurable increases in neutrophil kinetics. While several important parameters focusing on whole blood logistics are investigated here, additional variables related to the physiological factors in the in vivo environment are not discussed, including co-culture with common neighboring in vivo cell types (e.g., endothelial cells), oxygen tension (e.g., normoxia and hypoxia), and autologous signaling molecules (e.g., cytokines, hormones, growth factors). These factors will likely also in uence the overall quality of isolated neutrophils from whole blood. In addition to results reported above, inclusion of these parameters will lead to a more complete picture of the parameter space that is essential for maintaining in vivo-like neutrophil characteristics ex vivo to generate robust and reliable readouts of neutrophil behavior. These studies will be reported separately in a sister publication. We believe these efforts reported here provide a template for how other variables can be assayed to achieve a high-throughput, quality control method for sample analysis and measurement against non-speci c activation of neutrophils prior to interrogation in an ex vivo assay. Neutrophil isolation Neutrophils were isolated from whole blood using magnetic bead-based negative selection. Two negative selection kits -MACSxpress Whole Blood Neutrophil Isolation Kit, human (Miltenyi Biotec, and EasySep Direct Human Neutrophil Isolation Kit (STEMCELL, 19666) -were compared. In MACSxpress isolation, RBCs were further depleted from the neutrophil pellet using BD Pharm Lyse buffer (BD Biosciences, 555899), according to the manufacturer's instruction. In EasySep isolation, the neutrophil pellet was directly resuspended in appropriate media for further experiments. Cell count of each isolation was obtained using a hemocytometer (LW Scienti c).

Microscopic imaging
Bright-eld, uorescence images and videos were recorded on a Nikon Ti Eclipse inverted epi uorescence microscope (Nikon Instruments). The culture plate was kept at 37 o C, 21% or 5% O 2 , 5% CO 2 , and 95% RH in an onstage incubator (Bold Line, Okolab) during imaging or time lapse.

Area fraction analysis
The uorescence images of Sytox and Annexin V (4× magni cation) were batch processed and analyzed using a customized Java code (Github -https://github.com/jcai0791/Neutrophil-Project) calling functions from Fiji ImageJ (Supplementary Fig. 3). The work ow includes: 1) Open Fiji ImageJ. 2) Analyze → Set Measurements… (select "Area fraction"). 3) Open a set of the original .nd2 images with Sytox and Annexin V channels (Image → Type → 16-bit). 4) Image → Duplicate… (Separate the Sytox and Annexin V channels by making a duplicate). 5) Process → Subtract Background… (Rolling ball radius: 50 pixels). 6) Image → Adjust → Threshold… (pick a Threshold Method, Default for the Sytox channels, or MaxEntropy for Annexin V channels, select "Dark background"). 7) Analyze → Measure. 8) File → Save As → Jpeg… (use "default le name + Threshold Method-Sytox or Annexin V"). 9) Save Results (File → Save as… " le name.csv"). The data points in the .csv les were organized in the order of time points with conditions under each time point from the group of Sytox or Annexin V channels. For example, Correspondence and requests for materials should be addressed to Chao Li, or David J. Beebe.

Data availability
The data that support the ndings of this study are available from the corresponding authors upon reasonable request.

Code availability
All code used to analyze data and produce gures has been uploaded to Github    Table 2, Supplementary Fig. 1). The cell stages that Hoechst stains in H-S-A are highlighted by the blue-line box. The cell stages that Sytox stains in H-S-A are highlighted by bright green + olive green. The cell stages that Annexin V stains in H-S-A are highlighted by red + olive green. The inset microscopic images show the typical morphology/stains of neutrophil in different stages including healthy cell, early apoptosis, secondary necrosis, suicidal NETosis, and vital NETosis.

Figure 2
Whole blood logistics and ex vivo neutrophil interrogation in 2D cell culture. a A schematic visualizes the work ow of whole blood handling and culture conditions in this study. In a typical work ow, whole blood was collected from a donor at a designated location (e.g., hospital, clinic) and then transported to the lab for analysis. Before interrogation, the whole blood sample may be stored with different amounts of time,   Fig. 1a, b). The typical kinetic pro le consists of incubation phase (activation < 5%), exponential phase (fast activation), and stationary phase (saturated activation). Measurements of area fraction (%) for both b stationary storage and c rotary storage along with three culture conditions [standard, i.e., no oil (white dots), uorinated oil overlay (mint), silicone oil overlay (tan)] are not appreciably different in neutrophil activation but show less overall activation with silicone oil overlay ( Supplementary Fig. 7). Error bars are mean ± s.d..