B Cells BCR. Antigen. Igα Igβ ITAM. Activated B Cell. Germinal Center. B Cell. Follicular Helper T Cell. Plasmablast.

Transcription

1 s ntigen Igα Igβ ITM ctivated Germinal Center Follicular Helper T Cell Plasmablast Memory RnDSy-2945 Novus-2945

2 s lymphocytes ( cells) are an integral part of the humoral immune response due to their ability to produce antibodies against foreign antigens. cells originate from hematopoietic stem cells (HSCs) in the bone marrow, which is seeded during embryonic development by HSCs from the fetal liver. The initial stages of cell development are antigen-independent and involve the generation of several intermediary precursor cells that arise from lymphocyte progenitor cells including Pre-pro- cells, Pro- cells, and Pre- cells, which develop into immature cells. During these stages of development, cells undergo immunoglobulin gene rearrangement resulting in the expression of a mature cell receptor () that is capable of binding to antigen. This is followed by a selection process that involves editing or clonal deletion, designed to eliminate self-reactive immature cells. The majority of immature cells that survive this selection process leave the bone marrow and migrate to the spleen where they differentiate into transitional immature cells that then become immunocompetent naïve mature cells. Most naïve cells develop into follicular cells, while a small population becomes marginal zone cells (frequently called IgM memory cells in human). Following antigen-dependent activation, follicular cells participate in germinal center reactions where they differentiate into memory cells or long-lived, antibody-secreting plasma cells. While these developmental stages are similar for human cells and mouse conventional 2 cells, a second mouse cell lineage (1 cells) has also been described. 1 cells are abundant in the peritoneal cavities and can be further subdivided into 1a and 1b subsets. These along with other cell precursor cells and functionally distinct subtypes including marginal zone cells, follicular cells, memory cells, plasma cells, and regulatory cells can be distinguished from each other based on the expression of specific cell surface and intracellular markers. R&D Systems and Novus iologicals together offer the widest selection of fluorochrome-conjugated antibodies for detecting human and mouse cell subsets and further characterizing these cells. Cell Surface & Intracellular Markers Expressed at Different Stages of Development Cell Type Markers Markers Common Lymphoid CD10/Neprilysin + CD34 + Pax5 + Lin CD117/c-kit Sca-1/Ly6 Ly6D IL-7 Ra + Flt-3/Flk-2 + Lymphocyte CD10/Neprilysin + CD34 + Pax5 + Lin CD117/c-kit Sca-1/Ly6 Ly6D + IL-7 Ra + Flt-3/Flk Lin 220/CD45 R low + C1q R1/CD Lin 220/CD45 R + C1q R1/CD93 Pre-Pro CD117/c-kit low CD10/Neprilysin + CD34 + CD38 + Pax5 + Lin 220/CD45 R + CD24 low CD43 + C1q R1/CD93 + CD117/c-kit CXCR4 + Flt-3/Flk-2 + IL-7 Ra + IgM Pro- Pre- Immature CD117/c-kit low CD10/Neprilysin + + CD20/MS41 + CD24 + CD34 + CD38 + C1q R1/CD93 + IL-3 R + IL-7 Ra + Pax5 + Lin 220/CD45 R + + CD24 + CD43 + CD117/c-kit low IL-7 Ra + IgM CD117/c-kit CD10/Neprilysin + + CD20/MS41 + CD24 + CD34 CD38 + C1q R1/CD93 + IL-3 R + IL-4 Ra + IL-7 Ra + Pax5 + Lin 220/CD45 R + + CD24 + CD43 IL-7 Ra + IgM CD117/c-kit CD10/Neprilysin + + CD20/MS41 + CD21 + CD24 + CD27 220/CD45 R + + CD23/Fce RII CD24 + CD43 C1q R1/CD93 + CD38 + CD40 + C1q R1/CD93 + IL-4 Ra + IL-7 Ra IgD IgM + 1a Cell CD1d mid CD5 + high CD23/Fce RII CD43 + 1b Cell CD1d mid CD5 high CD23/Fce RII CD43 + Transitional CD10/Neprilysin low CD5 + + CD20/MS41 + CD21 + CD23/Fce RII + CD24 + CD27 CD38 + C1q R1/CD93 + TCI + T1: 220/CD45 R + + CD24 + CD43 C1q R1/CD93 + IgM + IgD low T2: 220/CD45 R + + CD24 + CD43 C1q R1/CD93 + IgM + IgD + Marginal Zone CD1c + + CD20/MS41 + CD21 + CD27 + FCRL3/FcRH3 + TCI + 220/CD45 R + CD1d + mid CD21 high CD23/Fce RII CD43 C1q R1/ CD93 IgM high IgD low Follicular ctivated Germinal Center CD10/Neprilysin + CD20/MS41 + CD21 + CD22/Siglec-2 + CD23/Fce RII + CD24 low CD27 CD38 low CXCR5 + TCI + MHC class II + 220/CD45 R + CD1d mid mid CD21 low CD23/Fce RII + CD43 CXCR5 + IgM low IgD high + CD20/MS41 + CD27 + CD38 + CD40 + CD83 + TCI + MHC class II + 220/CD45 R + + CD40 + MHC class II + Memory + CD20/MS41 + CD21 + CD27 mid/+ C1q R1/CD93 TCI + 220/CD45 R + + CD21 + CD27 mid/+ CD40 + MHC class II + Plasmablast CM + low CD27 high CD38 + C1q R1/CD93 + Syndecan-1/CD138 /low 220/CD45 R low + CD27 high CD38 + Syndecan-1/CD138 + CM + LIMP1 + low CD20/MS41 /low CD27 high CD38 high Syndecan-1/ CD138 + CXCR4 + MHC class II low 220/CD45 R low LIMP1 + CD27 high, CD38 low CXCR4 high Syndecan-1/CD138 + MHC class II /low Regulatory CD1d + CD5 + + CD21 + CD24 + IL-10 + IL-35 + TGF-b + CD1d + CD5 + + CD23/Fce RII /low CD24 + C1q R1/CD93 /low TIM-1 + IL-10 + IL-35 + TGF-b + Note: Lin for CLP, LP, 1 and 2 cells: CD3 CD4 CD8 Gr-1/Ly-6G Integrin am/cd11b TER-119 Lin for Pre-Pro, Pro-, Pre- cells: CD3 Gr-1/Ly- 6G Integrin am/cd11b TER-119 DOWNLOD NOW vailable as a reference tool.

3 Development Common Lymphoid Multipotent Cell Lymphocyte Pre-Pro Pro- Pre- Hematopoietic Stem Cell Pro- Pre- one Marrow Common Myeloid 1 2 Immature Mature Spleen 1a Transitional 1 Cell (mouse; from 1 Cell) T1 (mouse; from 2 Cell) 1b Peritoneal Cavity T2 T3 (human) Peripherial lood Naive Mature Long-lived Follicular Marginal Zone Memory ctivated ntigen ntigen ctivated Plasmablast Short-lived Follicular Helper T Cells ctivated Germinal Center Lymph Nodes and Mucosal-ssociated Lymphoid Tissue Germinal Center Memory Plasmablast Long-lived Peripherial lood

4 nalysis of Different Stages of Development in one Marrow by Flow Cytometry Multipotent Cell Common Lymphoid Lymphocyte Pre-Pro Pro- Pre- Hematopoietic Stem Cell Pro- Pre- Common Myeloid 1 2 Immature Mature one Marrow C Flt-3/Flk-2 CD117/c-kit Ly6D LP CLP Lineage cocktail Sca-1/Ly6 IL-7 Rα/CD127 Detection of Common Lymphoid Cells (CLPs) and Lymphocyte Cells (LPs) in one Marrow by Flow Cytometry. C57L/6 mouse bone marrow cells were depleted of CD3 +, CD4 +, CD8 +, Gr-1 +, CD11b/Integrin am +, TER cells by staining the cells with iotinylated nti- Monoclonal ntibodies against each of these markers followed by magnetic depletion using streptavidin-coated magnetic beads. Common lymphoid progenitor cells (CLPs: Lin /CD117 low /Sca-1 low /Flt-3 high /IL-7 Ra + /Ly-6D ) and lymphocyte progenitor cells (LPs: Lin /CD117 low /Sca-1 low /Flt-3 high /IL-7 Ra + /Ly-6D + ) in the remaining cell population were detected by staining the cells with () an lexa Fluor 405-conjugated lineage cocktail containing nti- Monoclonal ntibodies against each of the depletion markers and a PE-conjugated Rat nti- Flt-3/Flk-2 Monoclonal ntibody (R&D Systems, Catalog # F7681P). Lin /Flt-3 + cells were gated. () CD117 low /Sca-1 low cells in the Lin /Flt-3 + population were detected using an lexa Fluor 700-conjugated Rat nti- Sca-1/Ly6 Monoclonal ntibody (R&D Systems, Catalog # F1226N) and an PC-conjugated Rat nti- CD117/c-kit Monoclonal ntibody (R&D Systems, Catalog # F1356). (C) IL-7 Ra + /Ly-6D cells and IL-7 Ra + /Ly-6D + cells were detected in the Lin /Flt-3 + /CD117 mid/low /Sca-1 mid/low population using an lexa Fluor 488-conjugated Rat nti- IL-7 Ra/CD127 Monoclonal ntibody (R&D Systems, Catalog # F47742G) and a fl uorochrome-conjugated rat anti-mouse Ly-6D monoclonal antibody. C CD Lineage cocktail 220/CD45R 220/CD45R Detection of 1 and 2 Lymphocyte Cells in one Marrow by Flow Cytometry. () Lin /CD93 + and Lin /CD93 cells were detected in C57L/6 mouse bone marrow cells by staining with a lineage cocktail containing lexa Fluor 405-conjugated nti- Monoclonal ntibodies against CD3, CD4, CD8, CD11b/Integrin am, Gr-1, and TER-119 and an PC-conjugated Rat nti- C1q R1/CD93 Monoclonal ntibody (R&D Systems, Catalog # F1696). Lin /CD93 + cells and Lin /CD93 cells were gated. () 1 (Lin /CD93 + /220 low / + ) and (C) 2 (Lin /CD93 /220 + / ) progenitor cells were detected by staining with an lexa Fluor 488-conjugated Rat nti- 220/CD45 R Monoclonal ntibody (R&D Systems, Catalog # F1217G) and a PE-conjugated Rat nti- Monoclonal ntibody (Novus iologicals, Catalog # 24966).

5 Multipotent Cell Common Lymphoid Lymphocyt te Progenito or Pre-Pro Pro- Pre- Pro- Hematopoietic Stem Cell Pre- Common Myeloid 1 2 Immature Mature one Marrow Pro- and Pre-s 220/CD45R Pre-Pro s Lineage cocktail IgM C D Pre-s Pro-s CD24 Pre-Pro s CD24 CD43 Detection of Pre-Pro-, Pro-, and Pre-s in one Marrow by Flow Cytometry. () Lin /220 + cells were detected in C57L/6 mouse bone marrow by staining with a lineage cocktail containing lexa Fluor 405-conjugated nti- Monoclonal ntibodies against CD3, CD11b/Integrin am, Gr-1, and TER-119 and an lexa Fluor 750-conjugated Rat nti- 220/CD45 R Monoclonal ntibody (R&D Systems, Catalog # F1217S). () IgM / cells (pre-pro-) and IgM / + cells (pro- and pre-) were detected in the Lin / population by staining with a PE-Cy7-conjugated Rat nti- IgM Monoclonal ntibody (Novus iologicals, Catalog # 42940) and a PE-conjugated Rat nti- Monoclonal ntibody (Novus iologicals, Catalog # 24966). (C) Pre-pro- cells (Lin /220 + / /CD24 low /CD43 + /IgM ), (D) pro- cells (Lin /220 + / + /CD24 + /CD43 + /IgM ) and pre- cells (Lin /220 + / + /CD24 + /CD43 /IgM ) were detected by staining with an lexa Fluor 488-conjugated Rat nti- CD43 Monoclonal ntibody (Novus iologicals, Catalog # 43413F488) and an PC-conjugated Rat nti- CD24 Monoclonal ntibody (R&D Systems, Catalog # F8547). CD43

6 nalysis of Different Stages of Development in Spleen by Flow Cytometry Spleen 1a Transitional 1 Cell (mouse; from 1 Cell) T1 (mouse; from 2 Cell) 1b Peritoneal Cavity T2 T3 (human) Peripherial lood Naive Mature C T1 T2 Relative Cell Number CD43 IgM 220/CD45R Detection of Transitional 1 (T1) and Transitional 2 (T2) s in Splenocytes. () C57L/6 mouse splenocytes were stained with a PE-conjugated Rat nti- Monoclonal ntibody (Novus iologicals, Catalog # 24966) and + cells were gated. () /CD43 cells in the + cell population were detected by staining with an lexa Fluor 750-conjugated Rat nti- 220/CD45 R Monoclonal ntibody (R&D Systems, Catalog # F1217S) and an lexa Fluor 488-conjugated Rat nti- CD43 Monoclonal ntibody (Novus iologicals, Catalog # 43413F488). (C) Transitional 1 (T1) cells ( + /220 + /CD43-/IgM + /IgD low ) and transitional 2 (T2) cells ( + /220 + /CD43-/IgM + / IgD + ) were detected in the + /220 + /CD43- population by staining with an lexa Fluor 647-conjugated rat anti-mouse IgD monoclonal antibody and a PE-Cy7-conjugated Rat nti- IgM Monoclonal ntibody (Novus iologicals, Catalog # 42940). IgD CD23 CD5 1a 1b CD43 Detection of Transitional 1a and 1b Cells in Splenocytes. () C57L/6 mouse splenocytes were stained with an lexa Fluor 488-conjugated Rat nti- CD43 Monoclonal ntibody (Novus iologicals, Catalog # 43413F488) and an lexa Fluor 594-conjugated Rat nti- CD23/Fce RII Monoclonal ntibody (R&D Systems, Catalog # F6900T). CD43 + /CD23 cells were gated. CD1d mid cells in the CD43 + /CD23 population were detected by staining with an lexa Fluor 700-conjugated Rat nti- CD1d Monoclonal ntibody (Novus iologicals, Catalog # 43461F700; Data not shown). () Transitional 1a cells (CD5 + / high /CD1d mid /CD23 /CD43 + ) and transitional 1b cells (CD5 / high /CD1d mid / CD23 /CD43 + ) were detected in the CD1d mid /CD23 - /CD43 + population by staining with a PE-conjugated Rat nti- Monoclonal ntibody (Novus iologicals, Catalog # 24966) and an PC-conjugated Rat nti- CD5 Monoclonal ntibody (R&D Systems, Catalog # F115).

7 Spleen (human) Peripherial lood Naive Mature Long-lived Follicular Marginal Zone C MZ FO MZ CD43 CD1d CD23 Follicular CD21 Detection of Marginal Zone and Follicular -2 Cells in Splenocytes. () C57L/6 mouse splenocytes were stained with a PE-conjugated Rat nti- Monoclonal ntibody (Novus iologicals, Catalog # 24966) and an lexa Fluor 488-conjugated Rat nti- CD43 Monoclonal ntibody (Novus iologicals, Catalog # 43413F488). + / CD43 cells were gated. () Follicular -2 cells ( mid /CD1d mid /CD23 + /CD21 low /CD43 ) and marginal zone -2 cells ( mid /CD1d high /CD23 /CD21 high /CD43 ) were detected in the + /CD43 population by staining with a fl uorochrome-conjugated anti-mouse CD21 monoclonal antibody and an lexa Fluor 700-conjugated Rat nti- CD1d Monoclonal ntibody (Novus iologicals, Catalog # 43461F700). CD1d mid /CD21 low and CD1d high /CD21 high cells were gated. (C) Follicular -2 cells (CD1d mid /CD21 low ) and marginal zone -2 cells (CD1d high /CD21 high ) were stained for CD21 and CD23 using a fl uorochrome-conjugated anti-mouse CD21 monoclonal antibody and an lexa Fluor 594-conjugated Rat nti- CD23/ Fce RII Monoclonal ntibody (R&D Systems, Catalog # F6900T). CD21 C MZ FO Follicular CD23 IgM MZ CD43 CD21 Detection of IgD and IgM on Marginal Zone and Follicular -2 Cells from Splenocytes. () C57L/6 mouse splenocytes were stained with an lexa Fluor 488-conjugated Rat nti- CD43 Monoclonal ntibody (Novus iologicals, Catalog # 43413F488) and a PE-conjugated Rat nti- Monoclonal ntibody (Novus iologicals, Catalog # 24966). mid /CD43 cells were gated. () Follicular -2 cells ( mid /CD1d mid /CD23 + /CD21 low /CD43 ) and marginal zone -2 cells ( mid /CD1d high /CD23 /CD21 high / CD43 ) were detected in + /CD43 population by staining with a fl uorochrome-conjugated anti-mouse CD21 monoclonal antibody and an lexa Fluor 594-conjugated Rat nti- CD23/Fce RII Monoclonal ntibody (R&D Systems, Catalog # F6900T). CD21 low /CD23 + and CD21 high /CD23 cells were gated. (C) Expression of IgM and IgD on follicular -2 ( mid / CD23 + /CD21 low /CD43 /IgM low /IgD high ) and marginal zone -2 ( mid /CD23 /CD21 high /CD43 /IgM high /IgD low ) cells was detected using an lexa Fluor 647-conjugated rat anti-mouse IgD monoclonal antibody and a PE-Cy7-conjugated Rat nti- IgM Monoclonal ntibody (Novus iologicals, Catalog # 42940). IgD

8 nalysis of Memory s and Plasmablasts in Spleen by Flow Cytometry Spleen Long-lived ctivated ntigen ntigen ctiv C Memory Short-lived Plasmablast Follicular Helper T Cells ctivated Germinal Center Germinal Center Memory CD138 Plasmablast Detection of Memory s and Plasmablasts in Splenocytes. () Memory cells (CD27 + / + ) from immunized mouse alb/c splenocytes were detected by staining with an lexa Fluor 488-conjugated Rat nti- CD27/TNFRSF7 Monoclonal ntibody (Novus iologicals, Catalog # 11950F488) and a PEconjugated Rat nti- Monoclonal ntibody (Novus iologicals, Catalog # 24966). CD27 + / + cells were gated. () Plasmablasts ( + /CD27 + /CD38 + / CD138 + ) were detected in the CD27 + / + population by staining with an lexa Fluor 700-conjugated Rat nti- CD38 Monoclonal ntibody (Novus iologicals, Catalog # 77405F700) and an PC-conjugated Rat nti- CD138/Syndecan-1 Monoclonal ntibody (R&D Systems, Catalog # F2966). CD27 CD38 nalysis of Follicular Helper T Cells in Spleen CXCR5 Tfh Rat IgG 2 CD4 CD4 Detection of T Follicular Helper (Tfh) Cells in Splenocytes. () Immunized alb/c mouse splenocytes were stained an lexa Fluor 405-conjugated Rat nti- CD4 Monoclonal ntibody (R&D Systems, Catalog # F554V) and either () an PC-conjugated Rat nti- CXCR5 Monoclonal ntibody (Catalog # F6198) or () an PC-conjugated Rat IgG 2 Isotype Control (Catalog # IC006).

9 nalysis of s and Memory s by Flow Cytometry Spleen Long-lived ctivated ntigen ntigen ctivated Memory Short-lived Plasmablast Follicular Helper T Cells ctivated Germinal Center Germinal Center C s CD38 LIMP1 s CD3 CD20 CD38 D Memory s Detection of s and Memory s in Peripheral lood Mononuclear Cells by Flow Cytometry. Plasma cells (CD3 / low /CD20 /low /CD38 high /LIMP1 + ) in human peripheral blood mononuclear cells were detected by staining with () an lexa Fluor 405-conjugated nti- CD3e Monoclonal ntibody (R&D Systems, Catalog # F100V) and an lexa Fluor 594-conjugated nti- Monoclonal ntibody (R&D Systems, Catalog # F4867T). CD3 / + cells were gated. () Expression of CD20/MS41 and CD38 on cells in the CD3 / + gate was determined by staining with an PC-conjugated nti- CD20/MS41 Monoclonal ntibody (R&D Systems, Catalog # F4225) and a PerCP-conjugated nti- CD38 Monoclonal ntibody (R&D Systems, Catalog # F2404C). CD20 /low /CD38 + cells were gated. (C) Expression of LIMP1 in the CD20 /low /CD38 + cell population was determined by staining with a PerCP-conjugated nti- CD38 Monoclonal ntibody (R&D Systems, Catalog # F2404C) and a PE-conjugated nti- LIMP1/PRDM1 Monoclonal ntibody (R&D Systems, Catalog # IC36081P). (D) Memory cells were also detected in the starting population of human peripheral blood mononuclear cells by staining with a Fluorescein-conjugated nti- CD27/TNFRSF7 Monoclonal ntibody (R&D Systems, Catalog # F382F) and an lexa Fluor 594-conjugated nti- Monoclonal ntibody (R&D Systems, Catalog # F4867T). CD27 Pax5 Detection of Pax5 Expression in s by Flow Cytometry. peripheral blood mononuclear cells were surface stained using an PC-conjugated nti- Monoclonal ntibody (R&D Systems, Catalog # F4867). Cells were then fi xed and permeabilized and stained intracellularly using a Rabbit nti- Pax5 Monoclonal ntibody (R&D Systems, coming soon) followed by a PE-conjugated Goat nti-rabbit ntibody (R&D Systems, Catalog # F0110).

10 Fluorochrome-conjugated ntibodies for Detecting Different Stages of and Development ntibodies for Lineage Negative Markers used to Enrich for s Molecule Species Clone CD3 CD4 CD8a Integrin am/cd11b PC Fluorescein PE PerCP Fluorochrome-conjugated ntibodies for Flow Cytometry (Catalog #s) lexa Fluor UCHT1 F100 F100F F100P F100C F100G F100N 172 F4841 F4841F F4841P F4841C F4841G F4841N 145-2C PC 30149PE 30149PCP F484G dditional lexa Fluor conjugates /405/594/647/750 F484N F3791 F3791F F3791P F3791C F3791G F3791N RP-T PCP 27216F F700 GK1.5 F554 F554F F554P F554C F554G F554N F1509 F1509F F1509P F1509C F1509G F1509N C8/ PC RP-T PE PCP 25195PCP 34588F F F116 F116F F116P F116C F116G 34588F F700 F100V/F100T/F100R/ F100S F4841V/F4841T/ F4841R/F4841S F484U/F484V/F484T/ F484R/F484S 27216F405/ 27216F647 F554V/F554T/F554R/ F554S F1509V/F1509T/ F1509R/F1509S 34588F405/ 34588F F405/ 25195F647 F116V/F116T/F116R/ F116S iotin M M M116 ICRF44 F1699 F1699P F1699G M F16991 F16991P F16991C F16991G F16991N M1/70 F1124 F1124F F1124P F1124C F1124N F16991V/F16991T/ F16991R/F16991S F1124V/F1124T/ F1124R/F1124S M1124 Gr-1/Ly-6G R6-8C5 F1037 F1037F F1037P F1037C F1037N F1037V M1037 TER-119 TER-119 F1125 F1125F F1125P F1125N F1125V M1125 Unconjugated ntibodies (pplications) M100 (F, FC, ICC/IF, IP) M4841 (F, FC, ICC/IF, (FC); M484 (Depl, F, FC, IP) (/N, FC, IHC, IV) M554 (Depl, F, FC, M1509 (FC, ICC/IF) (FC, ICC/IF, IHC, IP, (FC, IHC, IV) M116 (Depl, F, FC, ICC/IF, IP) M1699 (FC, ICC/IF, IHC) M16991 (FC, ICC/IF) M1124 (FC, ICC/IF, M1037 (FC, ICC/IF, M1125 (FC, IHC, IP, ntibodies for Select Markers used to Identify Subsets by Flow Cytometry Molecule Species Clone PC Fluorescein PE PerCP Fluorochrome-conjugated ntibodies for Flow Cytometry (Catalog #s) lexa Fluor 220/CD45 R R3-62 F1217 F1217F F1217P F1217C F1217G F1217N 7-1/CD80 7-2/CD86 FF R/ TNFRSF13C CM/ TNFRSF17 LIMP1/ PRDM1 C1q R1/CD F140F F140P dditional lexa Fluor conjugates /594/647/750 F1217V/F1217T/ F1217R/F1217S F141 F141F F141P F141C F141N F141T/F141R GL1 F741 F741P F741C F741G Polyclonal F1162 F1162P Unconjugated ntibodies (pplications) M1217 (FC, ICC/IF, IP) M140 (/N, E, FC, IHC) (F, FC, M141 (/N, FC, M741 (/N, FC, F1162 (/N, FC, F1755F M1755 (FC) Polyclonal F193 F193F F193P F193N IC36081 IC36081P IC36081G IC36081N IC36081R / 3H2-E8 235PC 235F 235PE 235PCP 235F F F405/ 235F647 F193 (/N, E, FC, 235 (ChIP, E, FC, ICC/IF, IHC, F23791 M23791 (FC, F1696 F1696P M1696 (FC, ICC/IF) CD1c Polyclonal F5910 F5910P pplication Key: /N locking/neutralization ChiP Chromatin Immunoprecipitation Depl Depletion E ELIS F Functional ssay FC Flow Cytometry ICC/IF Immunocytochemistry/Immunofluorescence IHC Immunohistochemistry IP Immunoprecipitation IV In vitro W Western lot R&D Systems product Novus iologicals product

11 ntibodies for Select Markers used to Identify Subsets by Flow Cytometry CD1d CD5 Molecule Species Clone Neprilysin/ CD10 CD20/MS PC 43461PC PC Fluorescein PE PerCP 43460PE 43461PE F1636P Fluorochrome-conjugated ntibodies for Flow Cytometry (Catalog #s) 43460PCP 43461PCP lexa Fluor dditional lexa Fluor conjugates /594/647/ F F F F F115 F115F F115P F115N F115V HI10a 77917PC PE 77917PCP 77917F F700 4G7-2E3 F4867 F4867F F4867P F4867C F4867N LT19 C19 N PC 25196PC N PE G SJ25-C D N PCP 25196PCP 24965PCP N F F F488 N F F F F405/ 43460F F405/ 43461F F405/ 77917F647 F4867T/F4867R/ F4867S N F405/ N F F405/ 25196F F405/ 24965F647 Unconjugated ntibodies (pplications) (FC, (FC, M1636 (F, FC, M115 (F, FC, IHC, IP) (FC, M4867 (FC) N (FC, IP) (FC, ICC/IF, IHC, IV, (FC, ICC/IF) (FC, (FC) F4225 F4225F F4225P F4225N F4225V M4225 (FC) 2H7 IS PC 43435PC PE 43435PE 64858PCP 43435PCP 64858F F F F F405/ 64858F F405/ 43435F (FC, (FC, CD F4909 F4909F F4909P M4909 (FC, CD23/Fce RII CD24 CD25/IL-2 Ra CD F123P M123 (/N, FC, F6900 F6900P F6900T M6900 (FC) ML5 F5247 / M1/ PC PE 77388PCP 77388F F F405/ 77388F (FC) F8547 F8547P F8547G M8547 (FC, IHC) F1020 F1020P F1020G M1020 (FC, C (FC) F2438 F2438P F2438C F2438G M2438 (FC) PC D F382 F382F F382P / LG / /Rat LG.7F PC PE 27425PCP 43428PCP 27425F F F F F405/ 27425F F405/ 43428F (FC) M382 (/N, FC, (FC, (F, FC, IP) F5741 F5741P M5741 (E, FC) CD F229F F229P M229 (F, FC, CD34 CD38 QEnd10 F7227 F7227P F7227G F72271P F72271G F2404 F2404F F2404P F2404C HIT2 N N PC PE 77405PCP N F F488 N F F700 N F405/ N F F405/ 77405F647 M72271 (FC, ICC/ IF) M2404 (FC, ICC/IF, IP) N (FC, IHC, (FC)

12 Fluorochrome-conjugated ntibodies for Detecting Different Stages of and Development continued ntibodies for Select Markers used to Identify Subsets by Flow Cytometry Molecule Species Clone CD40/ TNFRSF5 CD43 CD117/c-kit CXCR4 CXCR5 PC Fluorescein PE PerCP F6321 F6321P 5C Fluorochrome-conjugated ntibodies for Flow Cytometry (Catalog #s) lexa Fluor dditional lexa Fluor conjugates /594/647/750 Unconjugated ntibodies (pplications) M6321 (F, FC, ICC/IF) (F, FC, IHC) F2038 F2038P M2038 (FC, ICC/IF) DF-T1 R2/ PC 43413PC 33140PE 33140PCP PE 43413PCP F332 F332P F332C / / D PC 765PC PE 765PE F1356 F1356P 77477PCP 765PCP 33140F F F F F F F F F405/ 33140F F405/ 43413F F405/ 77477F F405/ 765F (E, FC, ICC/IF, IHC, IP, (FC, IP, M332 (/N, E, FC, (FC, 765 (FC, ICC/ IF) M1356 (FC, IHC, 12G5 F170 F170F F170P F170C F170G F170N M170 (/N) F173 F173P F173C F173G F173N M173 (/N, FC) F21651 F21651F F21651P F21651C F190 F190F F190P F190C F190N M21651 (/N, FC, ICC/IF, IHC) M190 (/N, FC, ICC/IF, IHC) F6198 F6198F F6198P F6198C M6198 (FC, ICC/IF) FCRL3/FcRH F3126 F3126P F3126G M3126 (FC) Flt-3/Flk-2/ CD135 HL-DR HVEM/ TNFRSF14 IgD F812 F812F F812P F812N M812 (FC) F7681 F7681P M7681 (FC, ICC/IF) L203 F4869 F4869F F4869P F4869C F4869N L PC PE 77855PCP 77855F F700 F4869V/F4869T/ F4869R/F4869S 77855F405/ 77855F647 M4869 (FC) (FC, IHC, IP, F356 M356 (E, FC, 11-26c (FC) MS PC PE IgM II/ IL-3 Ra 48638PCP 48638F F F301 F301P F301C F301G F301N 6H6 1185PC PE 1185PCP 1185F F700 IL-4 Ra F230 F230F F230P F230C F230N IL-7 Ra/CD127 IL F405/ 48638F F405/ 1185F (FC) (E, FC, IHC) M301 (/N, FC, ICC/IF, IHC, 1185 (FC, IHC, M230 (/N, FC, IHC, F306 F306P F306C F306G F306N M306 (FC, 7R34 F47742 F47742P F47742G F47742N IC2172F IC2172P JES3-9D7 P-M PC 06673PE 06673PCP 06673F F F405/ 06673F (E, FC, (FC, IP) pplication Key: /N locking/neutralization ChiP Chromatin Immunoprecipitation Depl Depletion E ELIS F Functional ssay FC Flow Cytometry ICC/IF Immunocytochemistry/Immunofluorescence IHC Immunohistochemistry IP Immunoprecipitation IV In vitro W Western lot R&D Systems product Novus iologicals product

13 ntibodies for Select Markers used to Identify Subsets by Flow Cytometry Molecule Species Clone MHC class II TCI/ TNFRSF13 TGF-b1 CVS20 M5/ F6118 F6118F PC Fluorescein PE PerCP Fluorochrome-conjugated ntibodies for Flow Cytometry (Catalog #s) lexa Fluor dditional lexa Fluor conjugates /594/647/ F488 Unconjugated ntibodies (pplications) (FC, F1741 F1741P F1741C F1741G M1741 (/N, FC) F1041 F1041P M1041 (E, FC, 9016 IC240 IC240F IC240P M240 (/N, E, FC, IHC, M7666 (FC) TGF-b1, 2, 3 Multi-species 1D11 IC1835 IC1835P IC1835N Pax5 / 1H Sca-1/Ly F1226 F1226P F1226G F1226N M1226 (FC, ICC/IF) L-Selectin/ CD62L Siglec-2/CD F5761F F5761P M5761 (FC) MEL (FC, F1968F F1968P M1968 (FC, ICC/IF) F2296 F2296F F2296P M2296 (FC, Siglec-G F7775 M7775 (FC) Syndecan-1/ CD F2780 F2780F F2780P M2780 (FC, IHC) N (F, FC, ICC/IF, IHC, F2966 F2966F F2966P M2966 (FC, ICC/IF) Learn more rndsystems.com/bcells and novusbio.com/b-cell-research

14 dditional Products for Research In addition to the large selection of fluorochrome-conjugated antibodies that are offered by R&D Systems and Novus iologicals, R&D Systems offers several kits to simplify your cell research including Isolation and Expansion Kits, and recombinant proteins for cell culture and differentiation. We also offer a wide range of assays designed to provide insight into the cell-mediated immune response such as ELISpot Development Modules, Luminex Immunoglobulin Isotyping ssays, and ELIS Kits to quantify cell-secreted molecules. Isolation Kits R&D Systems MagCellect Isolation Kits are designed to isolate cells from a mononuclear cell suspension by negative selection. Using these kits, unwanted cells are magnetically tagged with a biotinylated antibody cocktail and streptavidin ferrofluid. The cell suspension is subsequently placed in a magnetic field, and the desired, untouched cell population is harvested by aspiration and immediately available for a variety of downstream applications. Isolation Kits Kit Catalog # MagCellect Isolation Kit MGH103 MagCellect Isolation Kit MGM204 MagCellect Rat Isolation Kit MGR % % 0.31% % 10 3 CD % CD % % % Enrichment of s using the MagCellect Isolation Kit. Ficolled human peripheral blood mononuclear cells before () and after () isolation of cells using the MagCellect Isolation Kit (Catalog # MGH103). Dot plots reflect double-staining of all viable cells using a Fluorescein-conjugated nti- Monoclonal ntibody (R&D Systems, Catalog # F4867F) and a PE-conjugated nti- CD14 Monoclonal ntibody (R&D Systems, Catalog # F3832P). Luminex Immunoglobulin Isotyping ssay nalyte Learn more rndsystems.com/magcellect Luminex Immunoglobulin Isotyping ssay This bead-based assay is designed to simultaneously quantify multiple mouse immunoglobulin isotype concentrations in cell culture supernatants, serum, or EDT plasma. The assay is designed for use with the Luminex MGPIX, Luminex 100, Luminex 200, or io-rad io-plex dual laser, flow-based sorting and detection analyzers. Polystyrene eads Catalog # Magnetic eads Catalog # IgG 1 LXIGM1 LXMIGMG1 IgG 2 LXIGM2 LXMIGMG2 IgG 2 LXIGM3 LXMIGMG3 IgG 3 LXIGM4 LXMIGMG4 Ig LXIGM5 LXMIGMG5 IgM LXIGM6 LXMIGMG6 Immunoglobulin Isotyping ase Kit LXIGM LXMIGMG Learn more rndsystems.com/luminex

15 Expansion Kit R&D Systems CellXVivo Expansion Kit contains all of the reagents necessary to expand a population of 10 7 human cells 3 5-fold. Expansion Kit Kit Catalog # CellXVivo Expansion Kit CDK Mean RFU Cells Cultured with Expansion Kit asal Media Expansion of s using the CellXVivo Expansion Kit. cells were isolated from peripheral blood mononuclear cells and cultured for five days using reagents included in the CellXVivo Expansion Kit (R&D Systems, Catalog # CDK005). cell expansion was measured with Resazurin (R&D Systems, Catalog # R002). Recombinant Proteins R&D Systems is the most referenced manufacturer of recombinant and natural proteins in the scientific literature. y maintaining stringent production and purification standards, we ensure that our proteins provide researchers with industry-leading bioactivity and lot-to-lot consistency. In addition to the more than 4,800 proteins that we manufacture under standard conditions, we also offer nimal-free and GMP-grade recombinant proteins as well as custom protein development services. Select Recombinant Proteins for Culture & Differentiation Molecule Species Catalog # 6420-CL CD40 Ligand 8230-CL 202-IL IL IL 204-IL IL ML 206-IL IL ML 217-IL IL ML 8879-IL IL ML Recombinant IL-2, IL-4, and IL-6 are also available as GMP-grade proteins. Relative Cell Number CD20 Detection of CD20 in s. cells were expanded for 5 days using reagents included in the Expansion Kit. The cells were labeled with a PEconjugated nti- CD20 Monoclonal ntibody (R&D Systems, Catalog # F4225P; filled histogram) or a PE-conjugated IgG 1 Isotype control (R&D Systems, Catalog # IC002P; open histogram). Learn more rndsystems.com/cellxvivo Learn more rndsystems.com/proteins

16 -related ELIS Kits R&D Systems offers complete Quantikine ELIS Kits and DuoSet ELIS Development Systems for detecting cell-secreted cytokines Select -related ELIS Kits Molecule Species Quantikine ELIS Catalog # DuoSet ELIS Catalog # D6050 DY206 IL-6 M6000 DY406 D1000 DY217 IL-10 M1000 DY417 D100 DY240 TGF-b1 M100 DY1679 DT00C DY210 TNF-a MT00 DY410 ELISpot Development Modules ELISpot Development Modules contain the basic components required to develop an ELISpot assay to detect IgE, IgG, or IgM-secreting cells. ELISpot Development Modules Kit Catalog # IgE ELISpot Development SEL001 IgG ELISpot Development SEL002 IgM ELISpot Development SEL003 IgG ELISpot Development SEL004 IgM ELISpot Development SEL005 Novus-2945 Global info@bio-techne.com bio-techne.com/find-us/distributors TEL North merica TEL Europe Middle East frica TEL +44 (0) China info.cn@bio-techne.com TEL +86 (21) bio-techne.com For research use or manufacturing purposes only. Trademarks and registered trademarks are the property of their respective owners. R 6670