Anti integrin β1 antibody

To assay the expression of the β1 integrin, iPSC-derived BMECs were differentiated, fixed in 1% paraformaldehyde, and stained using a 1:1,000 dilution of anti-β1 integrin antibody (catalog number NB100-63255; Abcam, Inc.) in 0.5% bovine serum albumin (BSA) in PBS overnight at 4°C. The following day, fixed cells were washed in 0.5% BSA in PBS twice and stained with anti-mouse IgG Alexa Fluor 488 antibody (catalog number A-11001; Life Technologies, Inc.) for 1 h at room temperature. Cells were then washed twice in 0.5% BSA in PBS and run on a BD Accuri C6 flow cytometry instrument for fluorescence-activated cell sorting (FACS) analysis.

To explore whether hPBMCs immobilization effects in alginate microcarriers were cell to matrix adhesion mediated, β1 integrins were blocked with an anti-β1 integrin antibody and pro-amyloidogenic and pro-inflammation cellular parameters were assessed. The hPBMCs were incubated with anti-β1 integrin antibody (Abcam, cat. no. ab24693, 1:200) in serum-free RPMI-1640 nutrient medium (Gibco, cat. no. 11875093) for 30 min at 37°C. Cells were then encapsulated in alginate microcarriers as described above and at hours 24 the pro-amyloidogenic and pro-inflammation parameters assays were performed.

HCAECs obtained from Sciencell (CA, USA) were cultured in RPMI 1640 medium supplemented with 10% FBS or 20% human sera, as previously described [10 (link)]. Briefly, HCAECs were cultured in RPMI 1640 medium containing 20% KD serum (KDS) or healthy children serum (HCS) for 6 h to establish a KD cell model. Subsequently, mRNA was extracted from the HCAECs for reverse transcription and quantitative real-time polymerase chain reaction (qRT-PCR) analysis of plexin C1 and integrin β1.
For stimulation with recombinant human Sema7A (rhSema7A) obtained from R&D Systems (USA), after the 6-hour treatment with human sera, HCAECs were washed with phosphate buffer solution (PBS) and the medium was replaced with RPMI 1640 medium containing 10% FBS. Subsequently, 10 µg/ml of rhSema7A was added to the medium, and the cells were incubated for 12 h. The culture supernatants were collected for ELISA analysis of TNF-α, IL-1β, IL-6, and IL-18 using the EliKineTM kits from Abbkine (China). Additionally, mRNA was extracted from the HCAECs for reverse transcription and subsequent qRT-PCR analysis of TNF-α, IL-1β, IL-6, and IL-18.
For the receptor blockade assay, anti-integrin β1 antibody and anti-plexin C1 antibody from Abcam (UK) were added to the medium for 1 h. Afterward, the cells were washed with PBS and stimulated with rhSema7A.