pAECs were collected from wild-type pig aortas, and cultured as previously described [36 (link)]. pAECs of passages 3 to 5 were used as stimulators for the mixed lymphocyte reaction (MLR). The sub-confluent pAECs were activated for 72h by co-culture with recombinant pIFN-γ (50ng/mL, R&D Systems, Minneapolis, MN). Activation of the cells was evaluated by staining with swine leukocyte antigen (SLA) class I (mouse anti-pig SLA class I, clone JM1E3, Serotec, Raleigh, NC) and SLA class II (DR) (mouse anti-pig SLA class II, clone 2E9/13, BD Biosciences, San Jose, CA) using flow cytometry [38 (link)].
Jm1e3
Manufactured by Bio-Rad
Sourced in United States
The JM1E3 is a laboratory instrument designed for cell sorting and analysis. It is capable of detecting and separating cells based on their physical and fluorescent characteristics. The core function of the JM1E3 is to provide researchers with a tool for analyzing and sorting cell populations in a research setting.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant pifn γ, by R&D Systems (1 mentions)
Ifn γ, by Bio-Rad (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Goat anti mouse igg, by BioLegend (1 mentions)
Ss03389945 m1, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated goat anti mouse igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
4 protocols using jm1e3
1
Activation of Porcine Aortic Endothelial Cells
2
Porcine Alveolar Epithelial Cell Activation
3
Evaluating SLA-I and SLA-II Silencing in ICCs
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To evaluate SLA‐I silencing effects from four donors per each group, non‐transduced (NT), transduced with a non‐specific shRNA (shNS) and SLA‐I silenced (shβ2M) ICCs‐derived cells were stained with anti‐SLA class I‐unconjugated antibody (JM1E3, Bio‐Rad) and goat anti‐mouse IgG conjugated with PE as a secondary antibody (Biolegend). Regarding SLA‐II silencing effect, NT, non‐specific transduced (shNS) and SLA‐II silenced (shCIITA) ICCs‐derived cells were stained with anti‐SLA class II DQ (K274.3G8)‐unconjugated antibody (Bio‐Rad) and goat anti‐mouse IgG conjugated with PE as a secondary antibody (Biolegend). All samples were then analysed by flow cytometry. Transduction efficiency was determined by the percentage of GFP‐positive cells and the mean fluorescence intensity (MFI) measured by the PE secondary antibody representing the level of SLA‐I or SLA‐II expression on the cell membrane.
4
Silencing of Porcine SLA Class I and II
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SLA class I and II downregulation was achieved by transduction with lentiviral vectors containing shRNA sequences targeting porcine β2m and CIITA. To evaluate the SLA class I and class II silencing effect, non-transduced, non-silenced (shNS), and silenced (shβ2m + shCIITA) PTECs were analyzed. The silencing effect was evaluated at mRNA levels by qRT-PCR and at protein levels by flow cytometry after stimulation with interferon-gamma (IFN-γ) (50 ng/mL) for 48 h.
Total RNA was isolated from three SLA-silenced PTEC lines and reversed transcribed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR was utilized to detect β2m, CIITA, and SLA-DRα transcript level expression using Taqman assays (Ss03391156_m1; Ss06941905_g1; Ss03389945_m1; all Thermo Fisher Scientific), respectively. GAPDH (Ss03375629_u1, Thermo Fisher Scientific) expression was used as an endogenous control.
SLA class I and class II protein expression was determined by flow cytometry. Cell staining was performed using an anti-SLA class I antibody (JM1E3; Bio-Rad Laboratories GmbH, Hercules, CA, USA) and anti-SLA class II DQ antibody (K274.3G8; Bio-Rad Laboratories GmbH) followed by APC-conjugated anti-mouse IgG1 (RMG1-1; Invitrogen) or AlexaFluor488-conjugated goat anti-mouse IgG secondary antibody (Invitrogen) staining.
Total RNA was isolated from three SLA-silenced PTEC lines and reversed transcribed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR was utilized to detect β2m, CIITA, and SLA-DRα transcript level expression using Taqman assays (Ss03391156_m1; Ss06941905_g1; Ss03389945_m1; all Thermo Fisher Scientific), respectively. GAPDH (Ss03375629_u1, Thermo Fisher Scientific) expression was used as an endogenous control.
SLA class I and class II protein expression was determined by flow cytometry. Cell staining was performed using an anti-SLA class I antibody (JM1E3; Bio-Rad Laboratories GmbH, Hercules, CA, USA) and anti-SLA class II DQ antibody (K274.3G8; Bio-Rad Laboratories GmbH) followed by APC-conjugated anti-mouse IgG1 (RMG1-1; Invitrogen) or AlexaFluor488-conjugated goat anti-mouse IgG secondary antibody (Invitrogen) staining.
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