bio rad cat  (Bio-Rad)

Journal: iScience

Article Title: Investigation of a fluorescent reporter microenvironment niche labeling strategy in experimental brain metastasis

doi: 10.1016/j.isci.2024.110284

Figure Lengend Snippet: CD206+ macrophages are the most abundant cell population within the sLP-mCherry+ fraction and are located outside of the metastatic niche in the breast-BrM model (A) Representative flow cytometry plots of the GFP-neg mCherry-neg and GFP-neg mCherry+ cell fractions in the breast-BrM model, showing the gating strategy used. Microglia-like cells were defined as CD45 low CD11b high , myeloid cells as CD45 high CD11b high , lymphoid cells as CD45 high CD11b-neg, and “stromal cells” as CD45-neg CD11b-neg. (B) Relative quantification of cell populations in the GFP-neg mCherry-neg and in the GFP-neg mCherry+ fractions in the breast-BrM metastasis model. Dashed lines indicate paired samples. Metastatic hemispheres from n = 8 mice. (C) Representative FCM gating strategy to identify neutrophils and the different macrophage subpopulations. Neutrophils were identified as CD11b+ Ly6G + events, monocytes as Ly6C+ CD11b+ Ly6G-neg events, and monocyte-derived macrophages (MDMs) as CD45 + CD49d+. Among CD49d-neg events, subdural border-associated macrophages (SD-BAMs) were identified as CD206+ cells, choroid plexus border-associated macrophages (CP-BAMs) as MHCII+, and microglia were identified as CD206-neg MHCII-neg events. Among the CD49d+ events, three subsets of MDMs were detected based on CD206 and MHCII expression. (D) Quantification of relative mCherry uptake by the different cell populations in the metastatic hemisphere in the breast-to-brain metastasis model as a percentage of the total number of cells in each indicated subset. Metastatic hemispheres from n = 4 mice. (E) Representative IF images of brain tissue in the breast-BrM model. i) The peri-metastatic area. Scale bar 50 μm. ii) A parenchymal area of brain tissue, considerably further than 500 μm from the tumoral lesion. Scale bar 50 μm. iii) Detail of the parenchymal area shown above in (ii). Scale bar 10 μm. White arrows in (i) and (ii) indicate mCherry+ CD206+ cells. (F) Relative quantification by IF image analysis of mCherry+ DAPI+ cells in the CD206+ and CD206-neg fractions, stratified based on the distance between the detected cells and the tumoral lesion. Dashed lines indicate paired samples. Metastatic hemispheres from n = 5 mice. Statistical analysis in (B) was performed using paired t test with Wilcoxon correction. Statistical analysis in (F) was performed using paired t test. Data are represented as mean ± SD. ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Article Snippet: FCM: CD49d BV421, rat monoclonal anti-mouse (clone R1-2), dilution 1:160 , BD Biosciences , Cat# 564397; RRID: AB_2738789.

Techniques: Flow Cytometry, Derivative Assay, Expressing

Journal: iScience

Article Title: Investigation of a fluorescent reporter microenvironment niche labeling strategy in experimental brain metastasis

doi: 10.1016/j.isci.2024.110284

Figure Lengend Snippet:

Article Snippet: FCM: CD49d BV421, rat monoclonal anti-mouse (clone R1-2), dilution 1:160 , BD Biosciences , Cat# 564397; RRID: AB_2738789.

Techniques: Recombinant, Fluorescence, Membrane, Virus, Plasmid Preparation, Software, Flow Cytometry, Microscopy, Adhesive

Journal: bioRxiv

Article Title: Epigenetic control of CD8 + T cell tissue homing and tissue resident memory T cell precursors by the histone methyltransferase SUV39H1

doi: 10.1101/2024.05.13.593994

Figure Lengend Snippet: A. Representative dot plots and quantification of CD44 and CD62L expression in CD8 + T cells from the indicated non lymphoid organs in conditional SUV39H1 KO (Cre+) or WT (Cre-) littermates. B. t-SNE plots of merged flow cytometry data gated on total CD8 + T cells from the liver of 5 conditional SUV39H1 KO (Cre+) and 5 WT littermates (Cre-) at steady state. See Supp Table 1. C. Representative dot plots and quantification of CD69 + CD103 - and CD49d + Ly6c - populations in CD44 + CD8 + T cells from liver of conditional SUV39H1 KO mice (Cre+) or WT littermates (Cre-) at steady state. D. Representative dot plots and quantification of intravascular staining negative (iv-) cells among lung CD8 + T cells and CD69 + CD103 - or CD49d + Ly6c - populations in iv-CD44 + CD8 + T cells from conditional SUV39H1 KO mice (Cre+) or WT littermates (Cre-) at steady state. Each dot represents a mouse, different shapes represent different experiments. * p<0.05 ; ** p<0.01 ; *** p< 0.001 ; **** p<0.0001 ; ns: not significant by multiple unpaired t test with Holm-Šídák correction ( A and B ) or Mann Whitney test ( C and D ).

Article Snippet: Cell suspensions were prepared in PBS and stained with Near IR (Fisher Scientific L34975) or Acqua LIVE/DEAD™ Fixable Cell Stain Kit (Fisher Scientific L34957) according to manufacturer’s instructions to exclude dead cells and then labelled in FACS buffer with the following antibodies: V500 Rat anti-Mouse CD4 (RM4-5, BD), eF450 CD5 Monoclonal Antibody (53-7.3, eBiosciences), FITC anti-CD8α (53-6.7, BD), PerCP-Cy5.5 CD8 α (53-6.7, BD), PE CD8 α Monoclonal Antibody (53-6.7, Ficher Scientific), BUV395 Rat Anti-Mouse CD8 β (H35-17.2, BD), APC anti-mouse CD25 Antibody (PC61, Biolegend), PerCP-Cy5.5 Rat Anti-Mouse CD44 (IM7, BD), APC Rat Anti-Mouse CD44 (IM7, BD), APC-R700 Rat Anti-Mouse CD44 (IM7, BD), eFluor 450 CD45.1 Monoclonal Antibody (A20, Fisher Scientific), PE Mouse Anti-Mouse CD45.1 (A20, BD), APC-Cy7 CD45.1 (A20, BD), PE Mouse Anti-Mouse CD45.2 (104, BD), APC Mouse anti-Mouse CD45.2 (104, BD), BV605 Hamster Anti-Rat/Mouse CD49a (Ha31/8, BD), BV786 Rat Anti-Mouse CD49d (R1-2, BD), PE/Cyanine7 anti-mouse CD49d Antibody (R1-2, Biolegend), PE-Cy7 Rat Anti-Mouse CD62L (MEL-14, BD), APC Rat Anti-Mouse CD62L (MEL-14, BD), APC/Cyanine7 anti-mouse CD62L Antibody (MEL-14, Biolegend), PerCP-Cy5.5 Hamster Anti-Mouse CD69 (H1.2F3, BD), PE-Cy7 Hamster Anti-Mouse CD69 (H1.2F3, BD), Brilliant Violet 605™ anti-mouse CD73 Antibody (TY/11.8, Biolegend), eFluor 450 CD103 Monoclonal Antibody (2E7, Fisher Scientific), eF660 CD127 (A7R34, eBiosciences), Brilliant Violet 711 anti-mouse CX3CR1 (SA011F11, Biolegend), BV711 CXCR3 (CXCR3-173, BD), Brilliant Violet 650™ anti-mouse Ly-6C Antibody (HK1.4, Biolegend), FITC anti-mouse Ly-6C (HK1.4, Biolegend), APC Ly-6C Monoclonal Antibody (HK1.4, eBiosciences), PE-CF594 Hamster Anti-Mouse KLRG1 (2F1, BD), BUV737 Hamster Anti-Mouse TCR β Chain (H57-597, BD), Alexa-Fluor®488 Hamster Anti-Mouse TCR β Chain (H57-597, BD), PE-Cy™7 Hamster Anti-Mouse TCR β Chain (H57-597, BD), PE-CF594 TCR β Chain (H57-597, BD), eFluor 450 TRCR V alpha 2 (B20.1, Invitrogen), PerCP/Cy5.5 anti-mouse TCR Vα2 (B20.1, Biolegend).

Techniques: Expressing, Flow Cytometry, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Epigenetic control of CD8 + T cell tissue homing and tissue resident memory T cell precursors by the histone methyltransferase SUV39H1

doi: 10.1101/2024.05.13.593994

Figure Lengend Snippet: A. Scheme of the experiment. B. Representative dot plots of WT (CD45.1-) and SUV39H1 KO (CD45.1+) OTI cells (Vα2+) in the indicated organs 33 days after adoptive transfer. C. Quantification of the ratio between SUV39H1 KO and WT OTI cells within each organ. D. Representative dot plot of intravascular (iv) staining ( left ) and expression of CD49d and Ly6c ( right ) in each population at day 34 post transfer in CD8 + T cells from lungs from Exp. 2 in C . E. Quantification of the percentage and absolute numbers of CD49d + Ly6c - cells among SUV39H1KO or WT OTI cells in the indicated organs from Exp 1 in C . Numbers represent the fold increase between KO and WT condition for each organ. F. Histogram of CD69 expression in iv-CD49d + Ly6c - CD8 + T cells and proportions of CD69+ cells among iv+ and iv-CD49d + Ly6c - CD8 + T cells in lungs from Exp. 4 in C . Numbers represent p values. Each dot represents a mouse, different shapes represent different experiments. * p<0.05 ; ** p<0.01 ; *** p< 0.001 ; **** p<0.0001 ; ns: not significant by one sample t test compared to 1 ( C ), multiple ratio paired t test with Holm-Šídák correction ( E ) and multiple paired t test ( F ).

Article Snippet: Cell suspensions were prepared in PBS and stained with Near IR (Fisher Scientific L34975) or Acqua LIVE/DEAD™ Fixable Cell Stain Kit (Fisher Scientific L34957) according to manufacturer’s instructions to exclude dead cells and then labelled in FACS buffer with the following antibodies: V500 Rat anti-Mouse CD4 (RM4-5, BD), eF450 CD5 Monoclonal Antibody (53-7.3, eBiosciences), FITC anti-CD8α (53-6.7, BD), PerCP-Cy5.5 CD8 α (53-6.7, BD), PE CD8 α Monoclonal Antibody (53-6.7, Ficher Scientific), BUV395 Rat Anti-Mouse CD8 β (H35-17.2, BD), APC anti-mouse CD25 Antibody (PC61, Biolegend), PerCP-Cy5.5 Rat Anti-Mouse CD44 (IM7, BD), APC Rat Anti-Mouse CD44 (IM7, BD), APC-R700 Rat Anti-Mouse CD44 (IM7, BD), eFluor 450 CD45.1 Monoclonal Antibody (A20, Fisher Scientific), PE Mouse Anti-Mouse CD45.1 (A20, BD), APC-Cy7 CD45.1 (A20, BD), PE Mouse Anti-Mouse CD45.2 (104, BD), APC Mouse anti-Mouse CD45.2 (104, BD), BV605 Hamster Anti-Rat/Mouse CD49a (Ha31/8, BD), BV786 Rat Anti-Mouse CD49d (R1-2, BD), PE/Cyanine7 anti-mouse CD49d Antibody (R1-2, Biolegend), PE-Cy7 Rat Anti-Mouse CD62L (MEL-14, BD), APC Rat Anti-Mouse CD62L (MEL-14, BD), APC/Cyanine7 anti-mouse CD62L Antibody (MEL-14, Biolegend), PerCP-Cy5.5 Hamster Anti-Mouse CD69 (H1.2F3, BD), PE-Cy7 Hamster Anti-Mouse CD69 (H1.2F3, BD), Brilliant Violet 605™ anti-mouse CD73 Antibody (TY/11.8, Biolegend), eFluor 450 CD103 Monoclonal Antibody (2E7, Fisher Scientific), eF660 CD127 (A7R34, eBiosciences), Brilliant Violet 711 anti-mouse CX3CR1 (SA011F11, Biolegend), BV711 CXCR3 (CXCR3-173, BD), Brilliant Violet 650™ anti-mouse Ly-6C Antibody (HK1.4, Biolegend), FITC anti-mouse Ly-6C (HK1.4, Biolegend), APC Ly-6C Monoclonal Antibody (HK1.4, eBiosciences), PE-CF594 Hamster Anti-Mouse KLRG1 (2F1, BD), BUV737 Hamster Anti-Mouse TCR β Chain (H57-597, BD), Alexa-Fluor®488 Hamster Anti-Mouse TCR β Chain (H57-597, BD), PE-Cy™7 Hamster Anti-Mouse TCR β Chain (H57-597, BD), PE-CF594 TCR β Chain (H57-597, BD), eFluor 450 TRCR V alpha 2 (B20.1, Invitrogen), PerCP/Cy5.5 anti-mouse TCR Vα2 (B20.1, Biolegend).

Techniques: Adoptive Transfer Assay, Staining, Expressing

Journal: bioRxiv

Article Title: Epigenetic control of CD8 + T cell tissue homing and tissue resident memory T cell precursors by the histone methyltransferase SUV39H1

doi: 10.1101/2024.05.13.593994

Figure Lengend Snippet: A. Scheme of the experiment. B. Representative dot plots and quantification of the normalized numbers of iv-CD44 + CD8 + T cells and the normalized percentages of CD49d + Ly6c - cells among iv-CD44 + CD8 + T cells in lungs 28 days post X31 infection ( Memory ). Values were normalized to the mean of X31-infected WT group for each experiment and then pooled. C. Representative dot plots and quantification of the number of iv-CD44 + CD8 + T cells in lungs 3 days post PR8 infection ( Recall ). D. Representative dot plots and quantification of the number of CD49d + Ly6c - and CD69 + CD103 - iv-CD44 + CD8 + T cells in lungs 3 days post PR8 infection ( Recall ). * p<0.05 ; ** p<0.01 ; *** p< 0.001 ; ns: not significant by ANOVA test with Šídák’s multiple comparisons test.

Article Snippet: Cell suspensions were prepared in PBS and stained with Near IR (Fisher Scientific L34975) or Acqua LIVE/DEAD™ Fixable Cell Stain Kit (Fisher Scientific L34957) according to manufacturer’s instructions to exclude dead cells and then labelled in FACS buffer with the following antibodies: V500 Rat anti-Mouse CD4 (RM4-5, BD), eF450 CD5 Monoclonal Antibody (53-7.3, eBiosciences), FITC anti-CD8α (53-6.7, BD), PerCP-Cy5.5 CD8 α (53-6.7, BD), PE CD8 α Monoclonal Antibody (53-6.7, Ficher Scientific), BUV395 Rat Anti-Mouse CD8 β (H35-17.2, BD), APC anti-mouse CD25 Antibody (PC61, Biolegend), PerCP-Cy5.5 Rat Anti-Mouse CD44 (IM7, BD), APC Rat Anti-Mouse CD44 (IM7, BD), APC-R700 Rat Anti-Mouse CD44 (IM7, BD), eFluor 450 CD45.1 Monoclonal Antibody (A20, Fisher Scientific), PE Mouse Anti-Mouse CD45.1 (A20, BD), APC-Cy7 CD45.1 (A20, BD), PE Mouse Anti-Mouse CD45.2 (104, BD), APC Mouse anti-Mouse CD45.2 (104, BD), BV605 Hamster Anti-Rat/Mouse CD49a (Ha31/8, BD), BV786 Rat Anti-Mouse CD49d (R1-2, BD), PE/Cyanine7 anti-mouse CD49d Antibody (R1-2, Biolegend), PE-Cy7 Rat Anti-Mouse CD62L (MEL-14, BD), APC Rat Anti-Mouse CD62L (MEL-14, BD), APC/Cyanine7 anti-mouse CD62L Antibody (MEL-14, Biolegend), PerCP-Cy5.5 Hamster Anti-Mouse CD69 (H1.2F3, BD), PE-Cy7 Hamster Anti-Mouse CD69 (H1.2F3, BD), Brilliant Violet 605™ anti-mouse CD73 Antibody (TY/11.8, Biolegend), eFluor 450 CD103 Monoclonal Antibody (2E7, Fisher Scientific), eF660 CD127 (A7R34, eBiosciences), Brilliant Violet 711 anti-mouse CX3CR1 (SA011F11, Biolegend), BV711 CXCR3 (CXCR3-173, BD), Brilliant Violet 650™ anti-mouse Ly-6C Antibody (HK1.4, Biolegend), FITC anti-mouse Ly-6C (HK1.4, Biolegend), APC Ly-6C Monoclonal Antibody (HK1.4, eBiosciences), PE-CF594 Hamster Anti-Mouse KLRG1 (2F1, BD), BUV737 Hamster Anti-Mouse TCR β Chain (H57-597, BD), Alexa-Fluor®488 Hamster Anti-Mouse TCR β Chain (H57-597, BD), PE-Cy™7 Hamster Anti-Mouse TCR β Chain (H57-597, BD), PE-CF594 TCR β Chain (H57-597, BD), eFluor 450 TRCR V alpha 2 (B20.1, Invitrogen), PerCP/Cy5.5 anti-mouse TCR Vα2 (B20.1, Biolegend).

Techniques: Infection

Journal: bioRxiv

Article Title: Epigenetic control of CD8 + T cell tissue homing and tissue resident memory T cell precursors by the histone methyltransferase SUV39H1

doi: 10.1101/2024.05.13.593994

Figure Lengend Snippet: A. Scheme of the experiment. B. Representative dot plots and quantification of the expression of CD69 and CD103 in SUV39H1-deficient and WT OTI cells 30 days post X31-OVA infection in lungs ( Memory ). C. Representative dot plots from lungs and quantification of the percentage of CD49d + Ly6c - cells among SUV39H1-deficient and WT OTI cells in the indicated organs and the percentage of CD69 + KLRG1 - or CD49a + CX3CR1 - cells among CD49d + Ly6c - iv-CD44 + OTI cells in lungs 9 days post X31-OVA infection ( Acute ). D. Scheme of the experiment. E. Gating strategy, representative dot plots and quantification of the percentage and numbers of WT and SUV39H1 KO OTI cells among CD69 + CD103 - and CD69 + CD103 + iv-CD44 + OTI cells in lungs 28 days post X31-OVA infection ( Memory ) and 27 days post adoptive transfer. p<0.05 ; ** p<0.01 ; *** p< 0.001 ; **** p<0.0001 ; ns: not significant by multiple ratio paired t test (B), (C) top and (E) and ratio pared t test (C) bottom. B, C and E : Representative data from 2 independent experiments.

Article Snippet: Cell suspensions were prepared in PBS and stained with Near IR (Fisher Scientific L34975) or Acqua LIVE/DEAD™ Fixable Cell Stain Kit (Fisher Scientific L34957) according to manufacturer’s instructions to exclude dead cells and then labelled in FACS buffer with the following antibodies: V500 Rat anti-Mouse CD4 (RM4-5, BD), eF450 CD5 Monoclonal Antibody (53-7.3, eBiosciences), FITC anti-CD8α (53-6.7, BD), PerCP-Cy5.5 CD8 α (53-6.7, BD), PE CD8 α Monoclonal Antibody (53-6.7, Ficher Scientific), BUV395 Rat Anti-Mouse CD8 β (H35-17.2, BD), APC anti-mouse CD25 Antibody (PC61, Biolegend), PerCP-Cy5.5 Rat Anti-Mouse CD44 (IM7, BD), APC Rat Anti-Mouse CD44 (IM7, BD), APC-R700 Rat Anti-Mouse CD44 (IM7, BD), eFluor 450 CD45.1 Monoclonal Antibody (A20, Fisher Scientific), PE Mouse Anti-Mouse CD45.1 (A20, BD), APC-Cy7 CD45.1 (A20, BD), PE Mouse Anti-Mouse CD45.2 (104, BD), APC Mouse anti-Mouse CD45.2 (104, BD), BV605 Hamster Anti-Rat/Mouse CD49a (Ha31/8, BD), BV786 Rat Anti-Mouse CD49d (R1-2, BD), PE/Cyanine7 anti-mouse CD49d Antibody (R1-2, Biolegend), PE-Cy7 Rat Anti-Mouse CD62L (MEL-14, BD), APC Rat Anti-Mouse CD62L (MEL-14, BD), APC/Cyanine7 anti-mouse CD62L Antibody (MEL-14, Biolegend), PerCP-Cy5.5 Hamster Anti-Mouse CD69 (H1.2F3, BD), PE-Cy7 Hamster Anti-Mouse CD69 (H1.2F3, BD), Brilliant Violet 605™ anti-mouse CD73 Antibody (TY/11.8, Biolegend), eFluor 450 CD103 Monoclonal Antibody (2E7, Fisher Scientific), eF660 CD127 (A7R34, eBiosciences), Brilliant Violet 711 anti-mouse CX3CR1 (SA011F11, Biolegend), BV711 CXCR3 (CXCR3-173, BD), Brilliant Violet 650™ anti-mouse Ly-6C Antibody (HK1.4, Biolegend), FITC anti-mouse Ly-6C (HK1.4, Biolegend), APC Ly-6C Monoclonal Antibody (HK1.4, eBiosciences), PE-CF594 Hamster Anti-Mouse KLRG1 (2F1, BD), BUV737 Hamster Anti-Mouse TCR β Chain (H57-597, BD), Alexa-Fluor®488 Hamster Anti-Mouse TCR β Chain (H57-597, BD), PE-Cy™7 Hamster Anti-Mouse TCR β Chain (H57-597, BD), PE-CF594 TCR β Chain (H57-597, BD), eFluor 450 TRCR V alpha 2 (B20.1, Invitrogen), PerCP/Cy5.5 anti-mouse TCR Vα2 (B20.1, Biolegend).

Techniques: Expressing, Infection, Adoptive Transfer Assay

Journal: bioRxiv

Article Title: Epigenetic control of CD8 + T cell tissue homing and tissue resident memory T cell precursors by the histone methyltransferase SUV39H1

doi: 10.1101/2024.05.13.593994

Figure Lengend Snippet: A. Scheme of the experiment. B. Representative dos plots of iv staining ( top ) and CD49d and Ly6c expression in SUV39H1 KO and WT iv-OTI cells ( bottom ) in the lungs of mice at 33 days post transfer. C. Individual tumor growth curves in each experimental group. Inset ratios show the number of mice that developed tumors at the end of the experiment over the total number of mice in each experimental group (cut off average radiance = 2×10 p/s/cm /sr). Pooled of 2 independent experiments. D. Median tumor growth curves of SUV39H1 KO and WT OTI recipient mice in C . Each dot represents a mouse. * p<0.05; ns: not significant by unpaired t test ( B ) and mixed-effects model ( D ).

Article Snippet: Cell suspensions were prepared in PBS and stained with Near IR (Fisher Scientific L34975) or Acqua LIVE/DEAD™ Fixable Cell Stain Kit (Fisher Scientific L34957) according to manufacturer’s instructions to exclude dead cells and then labelled in FACS buffer with the following antibodies: V500 Rat anti-Mouse CD4 (RM4-5, BD), eF450 CD5 Monoclonal Antibody (53-7.3, eBiosciences), FITC anti-CD8α (53-6.7, BD), PerCP-Cy5.5 CD8 α (53-6.7, BD), PE CD8 α Monoclonal Antibody (53-6.7, Ficher Scientific), BUV395 Rat Anti-Mouse CD8 β (H35-17.2, BD), APC anti-mouse CD25 Antibody (PC61, Biolegend), PerCP-Cy5.5 Rat Anti-Mouse CD44 (IM7, BD), APC Rat Anti-Mouse CD44 (IM7, BD), APC-R700 Rat Anti-Mouse CD44 (IM7, BD), eFluor 450 CD45.1 Monoclonal Antibody (A20, Fisher Scientific), PE Mouse Anti-Mouse CD45.1 (A20, BD), APC-Cy7 CD45.1 (A20, BD), PE Mouse Anti-Mouse CD45.2 (104, BD), APC Mouse anti-Mouse CD45.2 (104, BD), BV605 Hamster Anti-Rat/Mouse CD49a (Ha31/8, BD), BV786 Rat Anti-Mouse CD49d (R1-2, BD), PE/Cyanine7 anti-mouse CD49d Antibody (R1-2, Biolegend), PE-Cy7 Rat Anti-Mouse CD62L (MEL-14, BD), APC Rat Anti-Mouse CD62L (MEL-14, BD), APC/Cyanine7 anti-mouse CD62L Antibody (MEL-14, Biolegend), PerCP-Cy5.5 Hamster Anti-Mouse CD69 (H1.2F3, BD), PE-Cy7 Hamster Anti-Mouse CD69 (H1.2F3, BD), Brilliant Violet 605™ anti-mouse CD73 Antibody (TY/11.8, Biolegend), eFluor 450 CD103 Monoclonal Antibody (2E7, Fisher Scientific), eF660 CD127 (A7R34, eBiosciences), Brilliant Violet 711 anti-mouse CX3CR1 (SA011F11, Biolegend), BV711 CXCR3 (CXCR3-173, BD), Brilliant Violet 650™ anti-mouse Ly-6C Antibody (HK1.4, Biolegend), FITC anti-mouse Ly-6C (HK1.4, Biolegend), APC Ly-6C Monoclonal Antibody (HK1.4, eBiosciences), PE-CF594 Hamster Anti-Mouse KLRG1 (2F1, BD), BUV737 Hamster Anti-Mouse TCR β Chain (H57-597, BD), Alexa-Fluor®488 Hamster Anti-Mouse TCR β Chain (H57-597, BD), PE-Cy™7 Hamster Anti-Mouse TCR β Chain (H57-597, BD), PE-CF594 TCR β Chain (H57-597, BD), eFluor 450 TRCR V alpha 2 (B20.1, Invitrogen), PerCP/Cy5.5 anti-mouse TCR Vα2 (B20.1, Biolegend).

Techniques: Staining, Expressing

Journal: bioRxiv

Article Title: Identification of essential modules regulating T cell migration to the central nervous system in multiple sclerosis

doi: 10.1101/2022.06.17.496548

Figure Lengend Snippet: A, Experimental designs. (1.) Single gene target KO-EGFP + T MBP cells were generated by CRISPR RNP editing, then co-transferred at a 1:1 ratio with control BFP + cells into a single animal. After three days, the relative proportions of control and KO cells in blood and CNS tissues were assessed by flow cytometry. (2.) To assess the role of the gene candidates in EAE pathology, either control or single gene KO T MBP cells were transferred into Lewis rats, and the disease score and body weight were evaluated over the following eight days. B, Representative flow cytometry plots of T MBP cells recovered from blood, meninges and parenchyma after a co-transfer experiment with Itga4 -KO and control cells. C, Migratory phenotype of Itga4 -KO cells compared to control, shown as the ratio of Itga4 -KO cell numbers/control cell numbers in meninges (left) or parenchyma (right) divided by the KO/control ratio in blood. A ratio of 1 indicates the migration behaviour of control T MBP cells, a ratio below 1 indicates impaired migration into the CNS. n = 4 rats. D, EAE score (bars) and weight changes (line) of Itga4 -KO-injected and control-cell-injected animals; n = 3 rats per group. E, Representative flow cytometry plots showing in grey the isotype-matched antibody labelling control, in lilac the control T MBP cells, and in green the Hsp90b1 -KO T MBP cells, for CD49d ( Itga4 ), CD11a ( ItgaL ) and CD18 ( Itgb2 ). F , Quantification of the integrin labelling median fluorescence intensity of the Hsp90b1 -KO cells, normalized to the control intensity. A ratio of 1 indicates the surface integrin expression levels of control T MBP cells, a ratio below 1 indicates reduced integrin surface expression. n = 8 ( Hsp90b1 -KO CD49d and CD11a), n = 4 ( Hsp90b1 -KO CD18), all independent stainings. G , Representative flow cytometry plots of a T MBP co-transfer experiment with Hsp90b1 -KO and control cells. H, Migratory phenotype of Hsp90b1 -KO cells compared to control T MBP cells. n = 8 rats. I, EAE score (bars) and weight changes (line) of Hsp90b1 -KO-injected and control-cell-injected animals; n = 3 rats per group. J, Schematic representation of the adhesion module centred around α4-integrin. The effect of each member on T MBP cell migration to the CNS is color-coded based on the results of the parenchyma vs blood comparison of the CRISPR screens and stars indicate significance across at least three pairwise tissue comparisons (see Methods ). C, F, H, One sample t-test or Wilcoxon signed-rank test against hypothetical mean = 1; D, I , repeated measures two-way ANOVA (days three to eight for disease score ( D, F = 240.3, P = 0.0001; I, F = 66.13, P = 0.0012) and 0 to 8 for weight changes ( D, F = 47.1, P = 0.0024; I , F = 11.53, P = 0.0274)) and Sidak’s multiple comparison test. Figures show mean ± s.d, P > 0.05 ns (non-significant), P < 0.05 *, P < 0.01 **, P < 0.001 ***, P < 0.0001 ****.

Article Snippet: The following antibodies were used, Mouse IgG1 Isotype control (Sigma M-1398, clone MOPC31c), mouse anti-rat CD49d (Thermo Fisher MA49D7, clone TA-2), mouse anti-rat CD11a (Biolegend 201902, clone wt.1), mouse anti-rat CD18 (Thermo Fisher MA1817, clone wt.3), APC conjugated Donkey anti-mouse IgG (Jackson ImmunoResearch 715-136-151, polyclonal).

Techniques: Generated, CRISPR, Flow Cytometry, Migration, Injection, Fluorescence, Expressing