Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Mass Cytometry Assessment of Cell Phenotypes and Signaling States in Human Whole Blood

doi: 10.1007/978-1-0716-2553-8_10

Figure Lengend Snippet: Whole blood phosphoflow panel 1

Article Snippet: 150 Nd , pStat5 [Y694] , 47 , Fluidigm 3150005A.

Techniques:

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Mass Cytometry Assessment of Cell Phenotypes and Signaling States in Human Whole Blood

doi: 10.1007/978-1-0716-2553-8_10

Figure Lengend Snippet: Whole blood phosphoflow panel 1

Article Snippet: 142 Nd cCasp3 D3E9 Fluidigm 3142004A 143 Nd CD19 HIB19 Biolegend 302202 144 Nd pPLCg2 [Y759] K86-689.37 Fluidigm 3144015A 145 Nd CD4 RPA-T4 Fluidigm 3145001B 146 Nd IgD IA6-2 Fluidigm 3146005B 147 Sm CD20 2H7 Fluidigm 3147001B 148 Nd IgA Polyclonal Fluidigm 3148007B 149 Sm CD25 2A3 Fluidigm 3149010B 150 Nd pStat5 [Y694] 47 Fluidigm 3150005A 151 Eu CD123 6H6 Fluidigm 3151001B 153 Eu pStat1 [Y701] 4a Fluidigm 3153005A 154 Sm CD45 HI30 Fluidigm 3154001B 155Gd CD27 L128 Fluidigm 3155001B 156 Gd p38 [T180/Y182] D3F9 Fluidigm 3156002A 157 Gd CD24 ML-5 Biolegend 311102 158 Gd pStat3 [Y705] 4/P-Stat3 Fluidigm 3158005A 159Tb CD11c Bu15 Fluidigm 3159001B 160Gd CD14 M5E2 Fluidigm 3160001B 161 Dy CD141(BDCA-3) AD5-14H12 Miltenyi 130-090-694 162 Dy CD66b 80H3 Fluidigm 3162023B 163 Dy CD56 NCAM16.2 Fluidigm 3163007B 164 Dy IkBa L35A5 Fluidigm 3164004A 165 Ho pCREB [S133] 87G3 Fluidigm 3165009A 166 Er CD16 B73.1 Biolegend 360702 167 Er CD38 HIT2 Fluidigm 3167001B 168 Er CD8 SK1 Fluidigm 3168002B 169 Tm CD45RA HI100 Fluidigm 3169008B 170 Er CD3 UCHT1 Fluidigm 3170001B 171 Yb pERK1/2 [T202/Y204] D13.14.4E Fluidigm 3171010A 172 Yb Anti-Ki-67 B56 Fluidigm 3172024B 174 Yb HLA-DR L243 Fluidigm 3174001B 175Lu CD7 CD7-6B7 Biolegend 343102 176 Yb CD127/IL-7Ra P48-48 Novus Bio MAB306-100 209Bi CD11b/Mac-1 ICRF44 Fluidigm 3209003B Open in a separate window 1 Open channels are not shown but include Pd channels, Cd channel, Pt channels, 89Y,152Sm and 173Yb Whole blood phosphoflow panel1.

Techniques:

Journal: bioRxiv

Article Title: Aiolos modulates the T FH and CD4-CTL differentiation programs via reciprocal regulation of the Zfp831/TCF-1/Bcl-6 axis and CD25

doi: 10.1101/2022.05.18.492485

Figure Lengend Snippet: ( A ) Naïve WT C57BL/6 mice were infected intranasally with 40 PFU influenza (A/PR8/34; “PR8”) for 8 days. Single-cell suspensions were generated from the draining (mediastinal) lymph node, and analysis of Aiolos protein expression in the indicated populations was performed via flow cytometry. Data are compiled from 2 independent experiments (n = 6 ± s.e.m; **** P < 0.0001; unpaired Student’s t-test). ( B ) Analysis of the percentage of bulk PD-1 HI Cxcr5 HI (T FH ) populations generated during influenza infection in WT versus Ikzf3 -/- mice. Data are compiled from 5 independent experiments (n = 17 ± s.e.m; *** P < 0.001; unpaired Student’s t-test). ( C ) Analysis of the percentage of influenza nucleoprotein (NP)-specific PD-1 HI Cxcr5 HI (T FH ) populations generated in response to influenza infection. Following single-cell suspension, cells were stained with fluorochrome-labeled tetramers to identify NP-specific populations (n = 13-14 ± s.e.m; data are compiled from 4 independent experiments; **** P < 0.0001; unpaired Student’s t-test). ( D ) Analysis of the percentage of influenza nucleoprotein (NP)-specific Bcl-6 HI Cxcr5 HI (T FH ) populations generated in response to influenza infection (n = 13 ± s.e.m; data are compiled from 4 independent experiments; *** P < 0.001; unpaired Student’s t-test). ( E ) ELISA analysis of indicated serum antibody levels in ng/mL at 8 d.p.i. Data are compiled from 3 independent experiments (n = 11 ± s.e.m, ** P < 0.01, ***< 0.001; unpaired Student’s t-test).

Article Snippet: Membranes were blocked with 2% nonfat dry milk in 1X TBST (10 mM Tris [pH 8], 150 mM NaCl, 0.05% Tween-20), and detection of indicated proteins was carried out using the following antibodies: Aiolos (clone D1C1E, Cell Signaling, 1:20,000), Bcl-6 (clone K112, BD Biosciences, 1:500), pSTAT5(Y694/9) (clone BD Biosciences, 1:5000), STAT5 (clone (D206Y, Cell Signaling, 1:5000), β-Actin (GenScript, 1:15,000), T-bet (clone 4B10 Santa Cruz, 1:500), Eomes (Abcam.

Techniques: Infection, Generated, Expressing, Flow Cytometry, Staining, Labeling, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Aiolos modulates the T FH and CD4-CTL differentiation programs via reciprocal regulation of the Zfp831/TCF-1/Bcl-6 axis and CD25

doi: 10.1101/2022.05.18.492485

Figure Lengend Snippet: Naive WT or Aiolos-deficient ( Ikzf3 -/- ) CD4 + T cells were cultured under TH1-polarizing conditions for 3 days. ( A, C-E) Transcript analysis for the indicated genes was performed via qRT-PCR. Data are normalized to Rps18 control and presented relative to the WT sample for each gene. (n=9 ± s.e.m; **P < 0.01, ***P < 0.001, ****P < 0.0001; unpaired Student’s t-test; n.d., not detected). (B) Immunoblot analysis of the indicated proteins. β-actin serves as a control to ensure equivalent protein loading. A representative image from 3 independent experiments is shown. (F-H) Analysis of cytotoxic effector molecule production by flow cytometry. Cells were stimulated for 3h with PMA and Ionomycin in the presence of protein transport inhibitors. Cells were fixed and permeabilized and stained for the indicated proteins. (n=10 ± s.e.m; **P < 0.01, ***P < 0.001, ****P < 0.0001; unpaired Student’s t-test).

Article Snippet: Membranes were blocked with 2% nonfat dry milk in 1X TBST (10 mM Tris [pH 8], 150 mM NaCl, 0.05% Tween-20), and detection of indicated proteins was carried out using the following antibodies: Aiolos (clone D1C1E, Cell Signaling, 1:20,000), Bcl-6 (clone K112, BD Biosciences, 1:500), pSTAT5(Y694/9) (clone BD Biosciences, 1:5000), STAT5 (clone (D206Y, Cell Signaling, 1:5000), β-Actin (GenScript, 1:15,000), T-bet (clone 4B10 Santa Cruz, 1:500), Eomes (Abcam.

Techniques: Cell Culture, Quantitative RT-PCR, Control, Western Blot, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Aiolos modulates the T FH and CD4-CTL differentiation programs via reciprocal regulation of the Zfp831/TCF-1/Bcl-6 axis and CD25

doi: 10.1101/2022.05.18.492485

Figure Lengend Snippet: Naïve WT and Aiolos-deficient CD4 + T cells were cultured in the presence of T H 1 polarizing conditions for 3 days. (A) Immunoblot analysis of the indicated proteins. β-actin serves as a loading control. A representative image from 3 independent experiments is shown. (B-C) ATAC-seq analyses of T H 1 samples overlaid with published STAT5 ChIP-seq data. WT (light blue, top track) and Aiolos-deficient (red, middle track) samples are displayed as CPM-normalized Integrative Genomics Viewer (IGV) tracks (representative from 2 independent experiments). STAT5 ChIP-seq data alignment (dark blue) is shown in the bottom track (from GEO # GSE102317 (GSM2734684)). Regulatory regions of note are indicated by dashed boxes. Approximate locations of primers used to assess STAT5 enrichment by ChIP are indicated with gray arrows. ( D-E ) Naïve WT and Aiolos deficient CD4 + T cells were cultured in the presence of T H 1 polarizing conditions for 4 days. ChIP analysis was performed for STAT5 and an IgG control at the indicated regions. Data are normalized to total input sample. IgG values were subtracted from percent enrichment and data are displayed relative to the WT sample. (n = 4 ± s.e.m; *P <0.05; unpaired Student’s t-test).

Article Snippet: Membranes were blocked with 2% nonfat dry milk in 1X TBST (10 mM Tris [pH 8], 150 mM NaCl, 0.05% Tween-20), and detection of indicated proteins was carried out using the following antibodies: Aiolos (clone D1C1E, Cell Signaling, 1:20,000), Bcl-6 (clone K112, BD Biosciences, 1:500), pSTAT5(Y694/9) (clone BD Biosciences, 1:5000), STAT5 (clone (D206Y, Cell Signaling, 1:5000), β-Actin (GenScript, 1:15,000), T-bet (clone 4B10 Santa Cruz, 1:500), Eomes (Abcam.

Techniques: Cell Culture, Western Blot, Control, ChIP-sequencing

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Mass Cytometry Assessment of Cell Phenotypes and Signaling States in Human Whole Blood

doi: 10.1007/978-1-0716-2553-8_10

Figure Lengend Snippet: Whole blood phosphoflow panel 1

Article Snippet: 142 Nd cCasp3 D3E9 Fluidigm 3142004A 143 Nd CD19 HIB19 Biolegend 302202 144 Nd pPLCg2 [Y759] K86-689.37 Fluidigm 3144015A 145 Nd CD4 RPA-T4 Fluidigm 3145001B 146 Nd IgD IA6-2 Fluidigm 3146005B 147 Sm CD20 2H7 Fluidigm 3147001B 148 Nd IgA Polyclonal Fluidigm 3148007B 149 Sm CD25 2A3 Fluidigm 3149010B 150 Nd pStat5 [Y694] 47 Fluidigm 3150005A 151 Eu CD123 6H6 Fluidigm 3151001B 153 Eu pStat1 [Y701] 4a Fluidigm 3153005A 154 Sm CD45 HI30 Fluidigm 3154001B 155Gd CD27 L128 Fluidigm 3155001B 156 Gd p38 [T180/Y182] D3F9 Fluidigm 3156002A 157 Gd CD24 ML-5 Biolegend 311102 158 Gd pStat3 [Y705] 4/P-Stat3 Fluidigm 3158005A 159Tb CD11c Bu15 Fluidigm 3159001B 160Gd CD14 M5E2 Fluidigm 3160001B 161 Dy CD141(BDCA-3) AD5-14H12 Miltenyi 130-090-694 162 Dy CD66b 80H3 Fluidigm 3162023B 163 Dy CD56 NCAM16.2 Fluidigm 3163007B 164 Dy IkBa L35A5 Fluidigm 3164004A 165 Ho pCREB [S133] 87G3 Fluidigm 3165009A 166 Er CD16 B73.1 Biolegend 360702 167 Er CD38 HIT2 Fluidigm 3167001B 168 Er CD8 SK1 Fluidigm 3168002B 169 Tm CD45RA HI100 Fluidigm 3169008B 170 Er CD3 UCHT1 Fluidigm 3170001B 171 Yb pERK1/2 [T202/Y204] D13.14.4E Fluidigm 3171010A 172 Yb Anti-Ki-67 B56 Fluidigm 3172024B 174 Yb HLA-DR L243 Fluidigm 3174001B 175Lu CD7 CD7-6B7 Biolegend 343102 176 Yb CD127/IL-7Ra P48-48 Novus Bio MAB306-100 209Bi CD11b/Mac-1 ICRF44 Fluidigm 3209003B Open in a separate window 1 Open channels are not shown but include Pd channels, Cd channel, Pt channels, 89Y,152Sm and 173Yb Whole blood phosphoflow panel1.

Techniques: