Fig 2: MDA-MB-231 spheroid preservation analysis after several days of storage in oxygen-free and 4 °C storage in OptiPASS® medium culture. Three-day-old spheroids cultured in normal culture conditions, i.e., 37 °C, 5% CO2 in OptiPASS® medium culture were exposed to oxygen-free conditions at 4 °C for 5, 7, 14 or 30 days. After each storage time, spheroid recovery was analyzed in comparison to nonstored control spheroids. Samples’ integrity aspects and sizes (a) were studied with bright field microscopy and object size algorithm (Cytation™3MV, Gen5, BioTek®, M = 4X, scale bar = 1000 µm). Growth curve slopes were calculated using spheroids’ size values between the 4th and the 14th days of culture, reflecting recovery of proliferation capacities after storage step (b). Spheroid viability after storage was assessed by Live/Dead® tests showing viable cells (green fluorescence) and dead cells (red fluorescence) using Cytation™3MV equipped with GFP and IP fluorescence cubes (M = 4X, scale bar = 1000 µm) (c). Complementary to this, spheroid metabolic activity was quantified using resazurin tests (d). Otherwise, spheroid tumoral proliferation gradient analysis was carried out by ki67 immunostaining at D4, D7 and D14 and imaged with Texas Red filter (M = 10X, scale bar = 200 µm) on Cytation™3MV instrument (BioTek®). Ki67-positive nuclei (in orange) were identified among other nuclei (in blue) with adapted algorithm on Gen5 software (BioTek®, pictures showed for nonstored controls and spheroids stored for 7 days) (e). Global ki67-expression was quantified and compared between nonstored controls and 3-, 5- and 7-day-long cold and oxygen-free storage conditions in OptiPASS® medium (f). Finally, spheroid hypoxia level was studied using ROS-ID® kit staining at D3, D4 and D14 (g,h). Spheroids were imaged with Cytation™3MV equipped with Texas Red fluorescence cube (M = 4X, scale bar = 500 µm) (g). Hypoxia level was quantified by acquired fluorescent signal intensity with Gen5 software (BioTek®) (h). For each storage condition, significances compared to nonstored condition were indicated as ns (not significant; p > 0.05), ** p < 0.01, **** p < 0.0001, ***** p < 0.00001.

Fig 3: Effects of drop delay on the yield and purity of sorted cells and beads. The effect of drop delay on the yield of beads was tested. Each of the 10 puddles was collected using a drop delay setting with 0.1 intervals. Precisely 50 droplets were deposited into each puddle at different drop delay settings. (A) The effect of drop delay on the yield was tested with automatic and manually adjusted drop delays. The red line is the automatic one, and the blue line is the other one. (B) Map of the correlation between drop delay and yield. (C) Effect of long-term sorting-related drop delay deviation on yield. (D, E) The R-max method was used to assess the effect of drop delay on yield and purity. CS&T beads consist of 3 µm bright or mid and 2 µm dim beads. According to the R-max method reported, two beads of 3 µm bright or mid and central sheath catch (CSC) were sorted at the same time. After flow cytometry detection, the R-max value and purity of two kinds of beads were analyzed. (D) Dot plots of flow cytometry detection. (a) Dot plots of CS&T beads. (b–g) Dot plots of CSC, mid and bright beads were sorted with delay value 31.26 (b–d, red frame) and 30.96 (e–g, blue frame). 31.26 is the optimal value. 30.96 is the value of deviation. (F) The yield and purity of cells were assessed under manually adjusted conditions. (G) The estimated drop delay for different experiments of testing drop delay changes. Jurkat cells stained with CFSE and Hoechst 33342 were mixed at 1 : 1 with unstained cells to obtain a suspension (1 × 107 cells·mL-1). The double-positive cells were sorted into 1536-well plates. Then, each well of cells in the 1536-well plate was scanned with a BioTek Cytation 1 Cell Imaging Multi-Mode Reader and imaged individually. The data were analyzed by gen5 3.04 software (BioTek, Winooski, VT, USA). Data are presented as the mean ± SEM. n = 3. Each experiment was repeated at least three times. *P < 0.05, **P < 0.01, ***P < 0.001, NS: no significance, by one-way ANOVA.

Fig 4: EXO70H4 contributes to bacterium-induced callose deposits, but not ROS burst. a Callose deposits area was measured in each genotype after four- to five-week-old plants were syringe-infiltrated water as a mock control or with 1 × 108 CFU.mL- 1 of Pst DC3118, STm 14,028 s, or E. coli O157:H7. Results are shown as average of three to four biological replicate (n = 18 to 37 ± SE) and the experiment was repeated twice with similar results. Statistical difference between the means (wild type versus mutant plant) was calculated using the two-tailed Student’s t-test (** = p < 0.01; *** = p < 0.001; ns = non-significant). The pictures on the left are representative of each plant genotype per treatment. b Leaf discs of four- to five-week-old plants were incubated with control solution or 1 × 108 CFU.mL- 1 of boiled Pst DC3118, STm 14,028 s, or E. coli O157:H7. Results are shown as the average of luminescence per genotype in three independent biological replicates (n = 21 to 37 ± SE). Luminescence was recorded over 60 min using a Synergy™ H1 microplate reader and the Biotek Gen5 (Biotek) software. RLU = Relative Light Units

Fig 5: MDA-MB-231 (a,b) and SUM1315 (c,d) spheroid development with OptiPASS medium. (a,c) Spheroid size monitoring: spheroids formed with reference and OptiPASS medium were imaged after 2, 8 and 14 days of culture with Cytation3MV (BIOTEK). The mean size of each spheroid condition (n = 48) was calculated by the “cellular analysis” algorithm of Gen5.03 software (BIOTEK), bars = s.e.m. (b,d) Spheroids viability/mortality profile: after 7 and 14 days of culture, spheroids (n = 5) were harvested and incubated with live/dead fluorescent probes. Green signals correspond to Calcein-AM penetration in viable cells, and red signals correspond to ethidium homodimer-1 penetration in dead/necrotic cells. Observations were made with Cytation3MV with focal height fixed to the center of each spheroid (GFP and Texas Red filters, objective = 10×). * = p < 0.05, *** = p < 0.001, ***** = p < 0.00001, two-sided student’s t-test comparison.