The variable light chain and variable heavy chain sequences of a murine anti-BamA hybridoma were determined using 5′ RACE followed by sequencing of the PCR amplified products. cDNAs encoded for both variable domains were gene synthesized (Genewiz) and cloned into a mammalian surface display vector. This vector contains the light chain connect to heavy chain via an IRES sequence and has a glycosylphosphatidylinositol linkage signal (GPI anchor) derived from human decay-accelerating factor fused to the C-terminus of the heavy chain to enable surface display of the IgG29 (link). CHO cells that were transiently transfected with the cDNA encoding for the membrane-bound anti-BamA antibody were stained with streptavidin PE-conjugated biotinylated BamA protein antigen in 200 ul FACS staining buffer (PBS with 0.5% BSA and 2 mM EDTA) at a final concentration of 5 ug BamA/μl staining volume. Cells were stained for 15 min at 4 °C. After washing twice with PBS, cells were run on a BD LSRFortessa™ X-20 cell analyzer and data analyses was performed using FlowJo v.9.7.7 software.
Lsrfortessa x 20 cell analyzer
Manufactured by FlowJo
The LSRFortessa™ X-20 cell analyzer is a flow cytometry instrument designed for high-performance cell analysis. It features a 20-color detection system and can efficiently process and analyze a wide range of biological samples.
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3 protocols using lsrfortessa x 20 cell analyzer
1
Murine anti-BamA antibody surface display
2
Isolation and Characterization of Intestinal Epithelial Cells
3
Quantifying Cellular Uptake of miR-34a Nanoparticles
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Using a 12-well plate, 150,000 A549 cells were seeded for four treatment groups in triplicates. Cells were treated with PBS, BL NPs, and miR-34a-FITC NPs. A 2 mg/mL NP dose and an equivalent 300 pmol of miR-34a-FITC were transfected with Lipofectamine using forward transfection. After 24 h, the cells were washed with PBS, trypsinized, and then transferred to Eppendorf tubes. The cells were centrifuged at 2,000 rpm for 4 min and washed with PBS. The final pellet was resuspended in 300 μL of PBS and passed through filtered fluorescence-activated cell sorting (FACS) tubes. The intracellular FITC signal was quantified using the LSR Fortessa X-20 cell analyzer and FlowJo. To investigate the route of endocytosis, endocytosis inhibitors such as chlorpromazine (10 μg/mL; Sigma-Aldrich, St. Louis, MO), genistein (200 μM; Sigma-Aldrich, St. Louis, MO), and amiloride (1 mM; Sigma-Aldrich, St. Louis, MO) were used to pre-treat A549 cells. The cells were pre-treated with the inhibitors for 30 min and then incubated with miR-34a-FITC NPs (2 mg/mL) for 4 h. Flow cytometry was then performed to quantify cellular uptake. The same experimental setup was used for imaging the cells using fluorescence microscopy. However, the cells were treated with miR-34a-FITC NPs for 8 h.
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