Facsaria fusion 1 cell sorter, by BD - Product details

1

Thymic Epithelial Cell Isolation and Analysis

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For thymic epithelial cell analysis, single cell suspensions were generated by digesting thymic lobes with collagenase dispase (2.5mg/ml, Roche) and DNase 1 (40mg/ml Roche). CD45^- cells were enriched by the depletion of CD45⁺ cells using anti-CD45 beads and LS columns (Miltenyi Biotec). The following antibodies were used for TEC analysis: anti-CD45 clone 30-F11 (eBioscience), anti-EpCAM1 clone G8.8 (eBioscience), anti-Ly51 clone 6C3 (Biolegend), anti-MHCII clone M5/114.15.2 (eBioscience), anti-CD80 clone 16-10A1 (Biolegend), CD104 clone 346-11A (Biolegend), and anti-MHCI 28-14-8. Biotinylated UEA-1 (Vector laboratories) was detected using streptavidin PECy7 (eBioscience). Cells were analysed using a LSR Fortessa (Becton Dickinson) with data analysis carried out using Flowjo v10 (Becton Dickinson). For cell sorting, TEC subsets were identified using the antibodies above, and isolated using a FACS Aria Fusion 1 cell sorter (Becton Dickinson).
The sorting strategy for the different TEC subsets were as follows, Cxcl12^DsRed+ cTEC: CD45^-EpCAM1⁺UEA1^-Ly51⁺CXCL12^DsRed+; CXCL12^DsRed- cTEC: CD45^-EpCAM1⁺UEA1^-Ly51⁺CXCL12^DsRed-; mTEC^lo: CD45^-EpCAM1⁺UEA1⁺Ly51^-CD80^-MHCII^-; mTEC^hi: CD45^-EpCAM1⁺UEA-1^-Ly51⁺CD80⁺MHCII⁺; CD104⁺ mTEC^lo, CD45^-EpCAM1⁺UEA1⁺Ly51^-CD80^-MHCII^-CD104⁺; CD104^- mTEC^lo, CD45^-EpCAM1⁺UEA1⁺Ly51^-CD80^-MHCII^-CD104^-.

White A.J., Parnell S.M., Handel A., Maio S., Bacon A., Cosway E.J., Lucas B., James K.D., Cowan J.E., Jenkinson W.E., Hollander G.A, & Anderson G. (2023). Diversity in Cortical Thymic Epithelial Cells Occurs through Loss of a Foxn1-Dependent Gene Signature Driven by Stage-Specific Thymocyte Cross-Talk. The Journal of Immunology Author Choice, 210(1), 40-49.

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Isolation and Flow Cytometry Analysis of Thymic Epithelial Cells

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For TEC analysis, single-cell suspensions were generated by digesting thymic lobes with collagenase Dispase (2.5 mg/ml; Roche) and DNase 1 (40 mg/ml; Roche). CD45⁻ cells were enriched by the depletion of CD45⁺ cells using anti-CD45 beads and LS columns (Miltenyi Biotec). The following Abs were used for TEC analysis: anti-CD45 clone 30-F11 (eBioscience), anti-EpCAM1 clone G8.8 (eBioscience), anti-Ly51 clone 6C3 (BioLegend), anti–MHC II clone M5/114.15.2 (eBioscience), anti-CD80 clone 16-10A1 (BioLegend), CD104 clone 346-11A (BioLegend), and anti–MHC I 28-14-8. Biotinylated UEA-1 (Vector laboratories) was detected using streptavidin PECy7 (eBioscience). Cells were analyzed using a LSR Fortessa (Becton Dickinson) with data analysis carried out using FlowJo v10 (Becton Dickinson). For cell sorting, TEC subsets were identified using the earlier Abs and isolated using a FACSAria Fusion 1 cell sorter (Becton Dickinson). The sorting strategy for the different TEC subsets was as follows: Cxcl12^DsRed+ cTEC, CD45⁻EpCAM1⁺UEA1⁻Ly51⁺CXCL12^DsRed+; CXCL12^DsRed− cTEC, CD45⁻EpCAM1⁺UEA1⁻Ly51⁺CXCL12^DsRed−; mTEC^lo, CD45⁻EpCAM1⁺UEA1⁺Ly51⁻CD80⁻MHC II⁻; mTEC^hi, CD45⁻EpCAM1⁺UEA⁺Ly51⁻CD80⁺MHC II⁺; CD104⁺ mTEC^lo, CD45⁻EpCAM1⁺UEA1⁺Ly51⁻CD80⁻MHC II⁻CD104⁺; and CD104⁻ mTEC^lo, CD45⁻EpCAM1⁺UEA1⁺Ly51⁻CD80⁻MHC II⁻CD104⁻.

White A.J., Parnell S.M., Handel A., Maio S., Bacon A., Cosway E.J., Lucas B., James K.D., Cowan J.E., Jenkinson W.E., Hollander G.A, & Anderson G. (2023). Diversity in Cortical Thymic Epithelial Cells Occurs through Loss of a Foxn1-Dependent Gene Signature Driven by Stage-Specific Thymocyte Cross-Talk. The Journal of Immunology Author Choice, 210(1), 40-49.

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3

Isolation and Flow Cytometry Analysis of Thymic Epithelial Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

For TEC analysis, single-cell suspensions were generated by digesting thymic lobes with collagenase Dispase (2.5 mg/ml; Roche) and DNase 1 (40 mg/ml; Roche). CD45⁻ cells were enriched by the depletion of CD45⁺ cells using anti-CD45 beads and LS columns (Miltenyi Biotec). The following Abs were used for TEC analysis: anti-CD45 clone 30-F11 (eBioscience), anti-EpCAM1 clone G8.8 (eBioscience), anti-Ly51 clone 6C3 (BioLegend), anti–MHC II clone M5/114.15.2 (eBioscience), anti-CD80 clone 16-10A1 (BioLegend), CD104 clone 346-11A (BioLegend), and anti–MHC I 28-14-8. Biotinylated UEA-1 (Vector laboratories) was detected using streptavidin PECy7 (eBioscience). Cells were analyzed using a LSR Fortessa (Becton Dickinson) with data analysis carried out using FlowJo v10 (Becton Dickinson). For cell sorting, TEC subsets were identified using the earlier Abs and isolated using a FACSAria Fusion 1 cell sorter (Becton Dickinson). The sorting strategy for the different TEC subsets was as follows: Cxcl12^DsRed+ cTEC, CD45⁻EpCAM1⁺UEA1⁻Ly51⁺CXCL12^DsRed+; CXCL12^DsRed− cTEC, CD45⁻EpCAM1⁺UEA1⁻Ly51⁺CXCL12^DsRed−; mTEC^lo, CD45⁻EpCAM1⁺UEA1⁺Ly51⁻CD80⁻MHC II⁻; mTEC^hi, CD45⁻EpCAM1⁺UEA⁺Ly51⁻CD80⁺MHC II⁺; CD104⁺ mTEC^lo, CD45⁻EpCAM1⁺UEA1⁺Ly51⁻CD80⁻MHC II⁻CD104⁺; and CD104⁻ mTEC^lo, CD45⁻EpCAM1⁺UEA1⁺Ly51⁻CD80⁻MHC II⁻CD104⁻.

White A.J., Parnell S.M., Handel A., Maio S., Bacon A., Cosway E.J., Lucas B., James K.D., Cowan J.E., Jenkinson W.E., Hollander G.A, & Anderson G. (2023). Diversity in Cortical Thymic Epithelial Cells Occurs through Loss of a Foxn1-Dependent Gene Signature Driven by Stage-Specific Thymocyte Cross-Talk. The Journal of Immunology Author Choice, 210(1), 40-49.

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Facsaria fusion 1 cell sorter

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3 protocols using facsaria fusion 1 cell sorter

Thymic Epithelial Cell Isolation and Analysis

Isolation and Flow Cytometry Analysis of Thymic Epithelial Cells

Isolation and Flow Cytometry Analysis of Thymic Epithelial Cells

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