cd55 biotin  (R&D Systems)

Journal: bioRxiv

Article Title: Engineering Human Pluripotent Stem Cell Lines to Evade Xenogeneic Transplantation Barriers

doi: 10.1101/2023.06.27.546594

Figure Lengend Snippet: (A) Teratoma assay of H1, HM-KO, HM-KO lenti, and HM-KO AAVS cells in B6 mice. Teratomas >1mm in diameter were scored as detectable (top panel), while measurements of the teratoma sizes are depicted in the bottom panel. The dashed line at 1mm represents the limit of detection. Mean values + SEM are shown for each group of mice. Ten mice are represented per group from two independent experiments. For teratoma detection, ***p<0.001 and ****p<0.0001 by log-rank test. For teratoma growth, ****p<0.0001 by 2-way ANOVA with post-hoc Tukey’s multiple comparisons test. (B) Representative immunohistochemistry (IHC) staining of sections of HM-KO lenti and AAVS teratomas removed on day 102 post transplantation into B6 mice. Sections were imaged at 20x magnification with 100µm scale bars and 40x magnification with 50µm scale bars. Sections were stained with DAPI in combination with antibodies for human mitochondria (hMito) or mouse CD47, CD55, and CD59.

Article Snippet: Sections were incubated overnight with unconjugated goat anti-mouse/rat CD47 N-terminal IgV-like extracellular domain antibody (R&D Systems), biotin-conjugated anti-mouse CD55 REAfinity antibody (clone REA300, Miltenyi Biotec), unconjugated rabbit anti-mouse CD59a antibody (clone 108, SinoBiological), and AF594-conjugated anti-human mitochondria antibody (clone 113-1, Novus Biologicals) followed by a 1hr incubation with Alexa Fluor 647-conjugated AffiniPure donkey anti-goat IgG antibody (Jackson ImmunoResearch), Alexa Fluor 647-conjugated streptavidin (Jackson ImmunoResearch), or Alexa Fluor Plus 555-conjugated donkey anti-rabbit IgG antibody (Invitrogen).

Techniques: Immunohistochemistry, Transplantation Assay, Staining

Journal: bioRxiv

Article Title: Engineering Human Pluripotent Stem Cell Lines to Evade Xenogeneic Transplantation Barriers

doi: 10.1101/2023.06.27.546594

Figure Lengend Snippet: (A) Representative flow cytometry plots of complement deposition on Chinese hamster ovary (CHO) cells with or without the expression of CD55 or CD46. CHO cells were transfected with the human AAVS constructs containing either human CD55 (top row) or CD46 (bottom row). Cells were incubated with anti-CHO antibody followed by human C7-deficient serum and were stained for complement fragment deposition (C3c, C3d, C4c). (B-C) Quantification of complement deposition on cells expressing CD55 (B) or CD46 (C). Mean values ± SEM are shown for 3-7 replicates from two independent experiments. *p<0.05 and **p<0.01 by Student’s 2-tailed t-test. (D) Flow cytometry plot of K562s transfected with the human AAVS construct containing HLA-E and hCD55. Cells were transfected, placed under selection, and sorted to obtain a pure population for downstream NK cell degranulation assays. (E) Representative flow cytometry plots of NK cell degranulation assays utilizing unstimulated PBMCs, PBMCs mixed with K562s, and PBMCs mixed with HLA-E-expressing K562s. Degranulation is measured by the expression of CD107a. Populations shown have been gated on CD56 + NK cells. (F) NKG2A + NK cell degranulation assay. The frequency of CD107a-expressing cells of NKG2A + NK cells was analyzed from unstimulated PBMCs, PBMCs with K562s, and PBMCs with HLA-E + K562s. Mean values ± SEM are shown for 6 replicates from two independent experiments. ****p<0.0001 by Student’s 2-tailed t-test.

Article Snippet: Sections were incubated overnight with unconjugated goat anti-mouse/rat CD47 N-terminal IgV-like extracellular domain antibody (R&D Systems), biotin-conjugated anti-mouse CD55 REAfinity antibody (clone REA300, Miltenyi Biotec), unconjugated rabbit anti-mouse CD59a antibody (clone 108, SinoBiological), and AF594-conjugated anti-human mitochondria antibody (clone 113-1, Novus Biologicals) followed by a 1hr incubation with Alexa Fluor 647-conjugated AffiniPure donkey anti-goat IgG antibody (Jackson ImmunoResearch), Alexa Fluor 647-conjugated streptavidin (Jackson ImmunoResearch), or Alexa Fluor Plus 555-conjugated donkey anti-rabbit IgG antibody (Invitrogen).

Techniques: Flow Cytometry, Expressing, Transfection, Construct, Incubation, Staining, Selection, Degranulation Assay

Journal: bioRxiv

Article Title: Engineering Human Pluripotent Stem Cell Lines to Evade Xenogeneic Transplantation Barriers

doi: 10.1101/2023.06.27.546594

Figure Lengend Snippet: (A) Teratoma assay of H1, HM-KO, HM-KO lenti, and HM-KO AAVS cells in B6 mice. Teratomas >1mm in diameter were scored as detectable (top panel), while measurements of the teratoma sizes are depicted in the bottom panel. The dashed line at 1mm represents the limit of detection. Mean values + SEM are shown for each group of mice. Ten mice are represented per group from two independent experiments. For teratoma detection, ***p<0.001 and ****p<0.0001 by log-rank test. For teratoma growth, ****p<0.0001 by 2-way ANOVA with post-hoc Tukey’s multiple comparisons test. (B) Representative immunohistochemistry (IHC) staining of sections of HM-KO lenti and AAVS teratomas removed on day 102 post transplantation into B6 mice. Sections were imaged at 20x magnification with 100µm scale bars and 40x magnification with 50µm scale bars. Sections were stained with DAPI in combination with antibodies for human mitochondria (hMito) or mouse CD47, CD55, and CD59.

Article Snippet: The following mouse antibodies were purchased from Miltenyi Biotec: CD55 – biotin; Qa-1b (6A8.6F10.1A6) – APC; Qa-1b – biotin.

Techniques: Immunohistochemistry, Transplantation Assay, Staining

Journal: bioRxiv

Article Title: Engineering Human Pluripotent Stem Cell Lines to Evade Xenogeneic Transplantation Barriers

doi: 10.1101/2023.06.27.546594

Figure Lengend Snippet: (A) Representative flow cytometry plots of complement deposition on Chinese hamster ovary (CHO) cells with or without the expression of CD55 or CD46. CHO cells were transfected with the human AAVS constructs containing either human CD55 (top row) or CD46 (bottom row). Cells were incubated with anti-CHO antibody followed by human C7-deficient serum and were stained for complement fragment deposition (C3c, C3d, C4c). (B-C) Quantification of complement deposition on cells expressing CD55 (B) or CD46 (C). Mean values ± SEM are shown for 3-7 replicates from two independent experiments. *p<0.05 and **p<0.01 by Student’s 2-tailed t-test. (D) Flow cytometry plot of K562s transfected with the human AAVS construct containing HLA-E and hCD55. Cells were transfected, placed under selection, and sorted to obtain a pure population for downstream NK cell degranulation assays. (E) Representative flow cytometry plots of NK cell degranulation assays utilizing unstimulated PBMCs, PBMCs mixed with K562s, and PBMCs mixed with HLA-E-expressing K562s. Degranulation is measured by the expression of CD107a. Populations shown have been gated on CD56 + NK cells. (F) NKG2A + NK cell degranulation assay. The frequency of CD107a-expressing cells of NKG2A + NK cells was analyzed from unstimulated PBMCs, PBMCs with K562s, and PBMCs with HLA-E + K562s. Mean values ± SEM are shown for 6 replicates from two independent experiments. ****p<0.0001 by Student’s 2-tailed t-test.

Article Snippet: The following mouse antibodies were purchased from Miltenyi Biotec: CD55 – biotin; Qa-1b (6A8.6F10.1A6) – APC; Qa-1b – biotin.

Techniques: Flow Cytometry, Expressing, Transfection, Construct, Incubation, Staining, Selection, Degranulation Assay