apc cy7 conjugated streptavidin  (Thermo Fisher)

Journal: Nature Immunology

Article Title: β-Glucan reprograms neutrophils to promote disease tolerance against influenza A virus

doi: 10.1038/s41590-024-02041-2

Figure Lengend Snippet: a , Mice were treated with β-glucan, HSCs or progenitors and immune cells were assessed in the BM at days 2, 4 and 7 post-β-glucan treatment. b – e , Expansion of LKSs ( b ), MPPs ( c ), GMPs ( d ) and GPs ( e ) in the BM ( n = 5). f – h , Representative FACS plots ( f ), frequency ( g ) and total cell counts of neutrophils ( h ) in the BM ( n = 5). i , C57BL/6 (WT and Il1r − / − ) mice were treated with β-glucan. j – l , Neutrophils in the BM ( j ), blood ( k ) and lungs ( l ) at day 4 post-β-glucan treatment ( n = 5). Data are pooled from two individual experiments. m , Survival of Il1r − / − or WT mice after β-glucan treatment following IAV infection (lethal dose) at day 7 ( n = 10). Data are represented as mean ± s.e.m. Data were analyzed using one-way ANOVA followed by Tukey’s multiple-comparison test ( b – e , g and h ) or two-way ANOVA followed by Šidák’s multiple-comparison tests ( j – l ). Survival was monitored by a log(rank) test ( m ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Illustrations in a and i created using BioRender.com .

Article Snippet: For staining of LKSs, HSCs and MPPs, streptavidin–APC-Cy7 (eBioscience, 1:100), anti-c-Kit–APC (clone 2B8, eBioscience, 1:100), anti-Sca-1–PE-Cy7 (clone D7, eBioscience, 1:100), anti-CD150 eFluor-450 (clone mShad150, eBioscience, 1:100), anti-CD48-PerCP-eFluor-710 (clone HM48-1, BD Bioscience, 1:100), anti-Flt3–PE (clone A2F10.1, BD Bioscience, 1:100) and anti-CD34–FITC (clone RAM34, eBioscience, 1:100) were added and incubated at 4 °C for 30 min. For staining of myeloid and lymphoid progenitors, streptavidin–APC-Cy7 (eBioscience, 1:100), anti-c-Kit–APC (clone 2B8, eBioscience, 1:100), anti-Sca-1–PE-Cy7 (clone D7, eBioscience, 1:100), anti-CD34–FITC (clone RAM34, eBioscience, 1:100), anti-CD16/32-PerCP-eFluor-710 (clone 93, eBioscience, 1:100) and anti-CD127 BV786 (clone SB/199, BD Bioscience, 1:100) or anti-CD127-BV605 (clone A7R34, BioLegend, 1:100) were added and incubated at 4 °C for 30 min. For cMoPs and downstream progenitors: BM cells were incubated with biotin antibodies against lineage markers as mentioned above, except we included anti-Ly6G (clone 1 A8-Ly6G) to replace anti-Ly6C/6G at 4 °C for 30 min.

Techniques: Infection, Comparison

Journal: Nature Immunology

Article Title: β-Glucan reprograms neutrophils to promote disease tolerance against influenza A virus

doi: 10.1038/s41590-024-02041-2

Figure Lengend Snippet: a – c , Rag1 − / − mice ( a ) were infected with IAV lethal dose ( b ) and sublethal dose ( c ) at day 7 post-β-glucan treatment to assess survival ( n = 10). d , C57BL/6 (WT and Rag1 − / − ) mice were treated with β-glucan. e – h , Expansion of LKSs ( e ), MPPs ( f ), GMPs ( g ) and GPs ( h ) assessed in the BM on day 4 post-β-glucan treatment ( n = 5). i , j , Total cell counts of neutrophils in BM ( i ) and lungs ( j ) on day 4 post-β-glucan treatment ( n = 8). k , C57BL/6 (WT and Rag1 − / − ) mice infected with IAV infection on day 7 post-β-glucan. l , Total cell counts of neutrophils in the lungs at day 6 post-IAV infection ( n = 4). m , Mice were treated with β-glucan and after 4 d lungs subjected to MACSima imaging. n , Quantification of corresponding pulmonary immune cells from MACSima imaging ( n = 4). o , Quantification of RORγt CD4 + T cells after β-glucan treatment ( n = 5). p , Intravascular staining of RORγt GFP/GFP or RORγt WT/GFP at day 4 post β-glucan treatment. q , r , Total cell counts of neutrophils in the lung vasculature ( q ) and parenchyma ( r ) ( n = 4). s , RORγt GFP/GFP or RORγt WT/GFP mice infected with IAV (lethal dose) at day 7 post-β-glucan treatment to assess survival. Data are represented as mean ± s.e.m. Data were analyzed using two-tailed, unpaired Student’s t -test ( n ), one-way ANOVA followed by Tukey’s multiple-comparison test ( o ) and two-way ANOVA followed by Šidák’s multiple-comparison tests ( e – j , l , q and r ). Survival was monitored by a log(rank) test ( b , c and s ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. NS, not significant. Illustrations in a , d , k and p created using BioRender.com .

Article Snippet: For staining of LKSs, HSCs and MPPs, streptavidin–APC-Cy7 (eBioscience, 1:100), anti-c-Kit–APC (clone 2B8, eBioscience, 1:100), anti-Sca-1–PE-Cy7 (clone D7, eBioscience, 1:100), anti-CD150 eFluor-450 (clone mShad150, eBioscience, 1:100), anti-CD48-PerCP-eFluor-710 (clone HM48-1, BD Bioscience, 1:100), anti-Flt3–PE (clone A2F10.1, BD Bioscience, 1:100) and anti-CD34–FITC (clone RAM34, eBioscience, 1:100) were added and incubated at 4 °C for 30 min. For staining of myeloid and lymphoid progenitors, streptavidin–APC-Cy7 (eBioscience, 1:100), anti-c-Kit–APC (clone 2B8, eBioscience, 1:100), anti-Sca-1–PE-Cy7 (clone D7, eBioscience, 1:100), anti-CD34–FITC (clone RAM34, eBioscience, 1:100), anti-CD16/32-PerCP-eFluor-710 (clone 93, eBioscience, 1:100) and anti-CD127 BV786 (clone SB/199, BD Bioscience, 1:100) or anti-CD127-BV605 (clone A7R34, BioLegend, 1:100) were added and incubated at 4 °C for 30 min. For cMoPs and downstream progenitors: BM cells were incubated with biotin antibodies against lineage markers as mentioned above, except we included anti-Ly6G (clone 1 A8-Ly6G) to replace anti-Ly6C/6G at 4 °C for 30 min.

Techniques: Infection, Imaging, Staining, Two Tailed Test, Comparison