supplement mix c 39165  (PromoCell)

Journal: bioRxiv

Article Title: Plasmin, the product of tissue plasminogen activator (tPA) treatment for ischemic stroke, impairs human brain endothelial barrier integrity

doi: 10.64898/2026.05.27.728289

Figure Lengend Snippet: Initially D-VLC-AMC plasmin fluorescent substrate assays were conducted to identify a culture media that does not contain plasminogen system components, so that the effect of plasmin on hCMEC/D3 could be assessed in isolation. The standard EBM-2MV serum containing media for hCMEC/D3, contains plasmin, plasminogen and factors that inhibit plasmin activity, and hence cannot be used for this study (A). serum-free EBM and EBM + 2MV supplements do not contain plasmin, plasminogen or factors that inhibit plasmin activity (A); however, they do contain an insufficient level of protein (Sup Fig 2), which is not physiologically representative and furthermore is unlikely to support low quantities of plasmin protein in solution. The base media was therefore changed to AIM-V, which contains a higher level of protein. AIM-V + 2MV supplements does not contain plasmin or plasminogen, and does not inhibit plasmin activity, and hence is suitable for studying the direct effect of exogenous plasmin on hCMEC/D3 (C). The data presented are the mean ± SEM of three independent experiments. ECIS monitoring demonstrates that AIM-V-2MV serum-free media supports the maintenance of a stable functional hCMEC/D3 brain endothelial barrier, with similar properties to hCMEC/D3 layers cultured in EBM-2MV serum-free media (B, C, E, & F). Briefly hCMEC/D3 cells were seeded into ECIS 96W20IDF plates and cultured in EBM-2MV serum containing media for 48 hours to allow a stable barrier to form. Cells were then washed thrice with their respective future culture media (i.e. AIM-V-2MV serum-free media, EBM-2MV serum-free media or EBM-2MV serum containing media). Cells were then cultured in their respective media for a further 40 hours. Barrier properties were monitored in real-time throughout the experiment using ECIS. Ribbon plots represent the mean ± SEM of data from three independent experiments, each consisting of three technical replicates. Data was normalised before treatments were applied using vascr. Immunocytochemistry illustrates that hCMEC/D3 brain endothelial cells cultured in AIM-V-2MV serum-free media express the key junction associated molecules VE-cadherin (CD144), p -Catenin, Claudin-5, ZO-1 and PECAM-1 (CD31) (D). Briefly hCMEC/D3 cells were cultured in Ibidi 96 well plates in EBM-2MV serum containing media for 48 hours. Cells were then washed thrice with their respective future culture media (i.e. AIM-V-2MV serum-free media or EBM-2MV serum containing media) and cultured in their respective media for 16 hours. Cells were fixed with paraformaldehyde, probed with antibodies detecting junctional molecules, washed, and probed with respective fluorophore conjugated secondary antibodies and DAPI. Cells were imaged using an Operetta CLS. Images are representative of 9 fields from each of three independent experiments.

Article Snippet: AIM-V serum-free media was prepared by supplementing AIM-V media (ThermoFisher Scientific, 12055091) with 1X GlutaMAX (ThermoFisher Scientific, 35050061) and 2MV supplements (PromoCell, C-39221 with FCS omitted) and stored at −80°C.

Techniques: Isolation, Activity Assay, Functional Assay, Cell Culture, Immunocytochemistry

Journal: bioRxiv

Article Title: Plasmin, the product of tissue plasminogen activator (tPA) treatment for ischemic stroke, impairs human brain endothelial barrier integrity

doi: 10.64898/2026.05.27.728289

Figure Lengend Snippet: hCMEC/D3 cells were seeded into ECIS 96W20IDF plates and cultured in EBM-2MV serum containing media for 48 hours to allow a stable barrier to form. Cells were then washed thrice with AIM-V-2MV serum-free media and cultured for 16 hours in AIM-V-2MV serum-free media. Cells were washed thrice and cultured for 1 hour in fresh AIM-V-2MV serum-free media ± 640nM plasminogen. tPA was then added to the respective wells and cells cultured for a further 24 hours. Barrier properties were monitored in real-time throughout the experiment using ECIS. Ribbon plots represent the mean ± SEM of data from three independent experiments, each consisting of three technical replicates. Data was normalised 1hr before plasminogen was added, using vascr. Statistical analysis of this data was carried out at 4 and 12 hours after tPA addition; each treatment response was compared with vehicle control using two-way ANOVA with Dunnett’s test (* p <0.05, ** p <0.01).

Article Snippet: AIM-V serum-free media was prepared by supplementing AIM-V media (ThermoFisher Scientific, 12055091) with 1X GlutaMAX (ThermoFisher Scientific, 35050061) and 2MV supplements (PromoCell, C-39221 with FCS omitted) and stored at −80°C.

Techniques: Cell Culture, Control

Journal: bioRxiv

Article Title: Plasmin, the product of tissue plasminogen activator (tPA) treatment for ischemic stroke, impairs human brain endothelial barrier integrity

doi: 10.64898/2026.05.27.728289

Figure Lengend Snippet: hCMEC/D3 cells were seeded into ECIS 96W20IDF plates and cultured in EBM-2MV serum containing media for 48 hours to allow a stable barrier to form. Cells were then washed thrice with AIM-V-2MV serum-free media and cultured in this media for 16 hours. Media was removed and replaced with respective treatment in AIM-V-2MV serum-free media. Cells were cultured for 24 hours. Barrier properties were monitored in real-time throughout the experiment using ECIS. Ribbon plots represent the mean ± SEM of data from three independent experiments, each consisting of three technical replicates (A-D). Data were normalised 2 hours before the addition of treatment using vascr. Statistical analysis of this data was carried out at 4 and 12 hours after treatment addition; each treatment response was compared with vehicle control using two-way ANOVA with Dunnett’s test (* p <0.05, ** p<0.01, *** p < 0.001,, **** p < 0.0001) (E-H).

Article Snippet: AIM-V serum-free media was prepared by supplementing AIM-V media (ThermoFisher Scientific, 12055091) with 1X GlutaMAX (ThermoFisher Scientific, 35050061) and 2MV supplements (PromoCell, C-39221 with FCS omitted) and stored at −80°C.

Techniques: Cell Culture, Control

Journal: bioRxiv

Article Title: Plasmin, the product of tissue plasminogen activator (tPA) treatment for ischemic stroke, impairs human brain endothelial barrier integrity

doi: 10.64898/2026.05.27.728289

Figure Lengend Snippet: hCMEC/D3 cells were seeded into ECIS 96W20IDF plates and cultured in EBM-2MV serum containing media for 48 hours to allow a stable barrier to form. Cells were then washed thrice with AIM-V-2MV serum-free media and cultured in this media for 16 hours. Media was removed and replaced with respective treatment in AIM-V-2MV serum-free media. Cells were cultured for 24 hours. Barrier properties were monitored in real-time throughout the experiment using ECIS. Ribbon plots represent the mean ± SEM of data from three independent experiments, each consisting of three technical replicates (A-D). Data was normalised 1 hour before the addition of treatment using vascr. Statistical analysis of this data was carried out at 4 and 12 hours after treatment addition; each treatment response was compared with vehicle control using two-way ANOVA with Dunnett’s test (* p <0.05, *** p < 0.001).

Article Snippet: AIM-V serum-free media was prepared by supplementing AIM-V media (ThermoFisher Scientific, 12055091) with 1X GlutaMAX (ThermoFisher Scientific, 35050061) and 2MV supplements (PromoCell, C-39221 with FCS omitted) and stored at −80°C.

Techniques: Cell Culture, Control

Journal: bioRxiv

Article Title: Plasmin, the product of tissue plasminogen activator (tPA) treatment for ischemic stroke, impairs human brain endothelial barrier integrity

doi: 10.64898/2026.05.27.728289

Figure Lengend Snippet: hCMEC/D3 cells were seeded into Ibidi 96 well plates in EBM-2MV serum containing media for 48 hours. Cells were then washed thrice with AIM-V-2MV serum-free media and cultured in AIM-V-2MV serum-free media for 16 hours. Vehicle or 640nM plasmin was then added and cells were cultured for 4 or 12 hours. Cells were fixed with paraformaldehyde, probed with antibodies detecting junctional molecules (VE-cadherin (CD144), β-Catenin, Claudin-5, ZO-1 and PECAM-1 (CD31) ), washed, and detected with their respective fluorophore conjugated secondary antibodies and DAPI. Single planes of the wells were imaged using an Operetta CLS. The images shown are representative of 9 fields from each of three independent experiments (A). Vjunctr was then used to quantify the total fluorescence intensity and the fluorescence intensity in the junctional area for each junctional molecule (B & C). The bar charts represent the mean ± SEM of data from three independent experiments, each of which consisted of three technical replicates of nine randomly selected fields from each well. Significant differences between plasmin and vehicle control for each junctional molecule was analysed using two-way ANOVA with Tukey’s range test (*** p<0.001).

Article Snippet: AIM-V serum-free media was prepared by supplementing AIM-V media (ThermoFisher Scientific, 12055091) with 1X GlutaMAX (ThermoFisher Scientific, 35050061) and 2MV supplements (PromoCell, C-39221 with FCS omitted) and stored at −80°C.

Techniques: Cell Culture, Fluorescence, Control