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Cd45.2 apc clone 104

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CD45.2-APC (clone 104) is a fluorochrome-conjugated antibody that binds to the CD45.2 antigen, which is a protein tyrosine phosphatase expressed on the surface of most hematopoietic cells. This product is intended for use in flow cytometry applications.

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Lab products found in correlation

Dihydroethidium, by Merck Group (8 mentions) Ter119 apc cy7 clone ter 119, by BioLegend (7 mentions) Hoechst 33342, by Merck Group (7 mentions) Cd3ε pe cy7 clone 145.2c11, by BioLegend (2 mentions) Hemocytometer, by Teknova (2 mentions) K2edta treated tubes, by BD (1 mentions) 7 aad, by BioLegend (1 mentions) Dnase 1, by Solarbio (1 mentions) Ls004186, by Worthington (1 mentions) Facsaria 2 cell sorter, by BD (1 mentions)

Close spelling variants of this product

CD45-APC (97 citations) CD45.2 (clone 104, (29 citations) CD45.2 (104, (22 citations) CD45 (Clone 104) (8 citations) CD45.2-APC (7 citations) Anti-CD45.2-APC-Cy7 (clone 104; (3 citations)

11 protocols using cd45.2 apc clone 104

1

Quantifying Plasmodium Yoelii Parasitemia

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Mice were infected with Plasmodium yoelii 17XNL by intravenous injection of 1 × 105 infected red blood cells (RBCs) in 200 uL of saline prepared from frozen stock. Parasitemia (i.e. percentage of total infected RBCs) was evaluated by flow cytometry between days 5–30 post-infection (p.i.) via blood taken from the tails of infected mice. Approximately 5 µL of whole blood was diluted in 100 µL of PBS followed by fixation in 0.00625% glutaraldehyde. The samples were then stained: CD45.2-APC (clone 104; Biolegend, San Diego, CA), Ter119-APC/Cy7 (clone TER-119; Biolegend, San Diego, CA), dihydroethidium (MilliporeSigma, St. Louis, MO), and Hoechst 33342 (MilliporeSigma, St. Louis, MO). After staining, samples were resuspended in flow cytometry buffer and analyzed; RBCs were gated by Ter119+CD45.2− followed by gating the infected subpopulation on dihydroethidium+Hoechst 33342+ to find the percentage of infected RBCs.
Denny J.E., Powers J.B., Castro H.F., Zhang J., Joshi-Barve S., Campagna S.R, & Schmidt N.W. (2019). Differential Sensitivity to Plasmodium yoelii Infection in C57BL/6 Mice Impacts Gut-Liver Axis Homeostasis. Scientific Reports, 9, 3472.
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2

Quantification of Antigen-Specific CD8+ T Cells

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On day 20 after immunizations, whole blood was collected in K2EDTA treated tubes (BD Biosciences), treated with ACK lysis buffer (KD Medical), washed, resuspended in cold FACS buffer (PBS supplemented with 2% FBS and 50 μM dasatinib), and plated in a 96-well U-bottom plate. Next, the cells were centrifuged for 5 min at 1,500 rpm and resuspended in FACS buffer and incubated with Fc-block (anti-CD16/CD32, clone 2.4G2; Tonbo) for 15 min at 4°C, and stained with antibodies CD45.2 (APC; clone 104; BioLegend), CD3ε (PE/Cy7; clone 145.2C11; BioLegend), and CD8α (APC/Cy7; clone 53–6.7; Tonbo) for 1 h at 4°C. Cells were then washed 3x in FACS buffer and then stained for 2 h with 1.5 μg/mL of PE-labeled OVA 257–264 pOVA/H-2Kb tetramer prepared according to a previously reported procedure.59 (link) Cells were then washed 3x in FACS buffer, resuspended using FACS buffer supplemented with 1 μg/mL DAPI, and analyzed using an Amnis CellStream Luminex flow cytometer. Representative flow cytometry data and gating strategies for determining the frequency of SIINFEKL-specific CD8+ T cells are shown in Supplementary Figure 3.
Carson C.S., Becker K.W., Garland K.M., Pagendarm H.M., Stone P.T., Arora K., Wang-Bishop L., Baljon J.J., Cruz L.D., Joyce S, & Wilson J.T. (2022). A Nanovaccine for Enhancing Cellular Immunity via Cytosolic Co-Delivery of Antigen and PolyIC RNA. Journal of controlled release : official journal of the Controlled Release Society, 345, 354-370.
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3

Quantification of Antigen-Specific CD8+ T Cells

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Splenocytes were plated at 3 × 106 cells/well in a 96-well U-bottom plate. Next, the cells were centrifuged for 5 min at 1,500 rpm and resuspended in FACS buffer and incubated with Fc-block (anti-CD16/CD32, clone 2.4G2; Tonbo) for 15 min at 4°C, and then stained with antibodies CD45.2 (APC; clone 104; BioLegend), CD3ε (PE/Cy7; clone 145.2C11; BioLegend), and CD8α (APC/Cy7; clone 53–6.7; Tonbo) for 1 h at 4°C. Cells were then washed 3x in FACS buffer and then stained for 2 h with 1.5 μg/mL of PE-labeled pOVA/H-2Kb tetramer. Cells were then washed 3x in FACS buffer and analyzed as described above. Representative flow cytometry data and gating strategies for determining the frequency of SIINFEKL-specific CD8+ T cells are shown in Supplementary Figure 3.
Carson C.S., Becker K.W., Garland K.M., Pagendarm H.M., Stone P.T., Arora K., Wang-Bishop L., Baljon J.J., Cruz L.D., Joyce S, & Wilson J.T. (2022). A Nanovaccine for Enhancing Cellular Immunity via Cytosolic Co-Delivery of Antigen and PolyIC RNA. Journal of controlled release : official journal of the Controlled Release Society, 345, 354-370.
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4

Murine Malaria Infection Monitoring

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C57BL/6N mice were injected intravenously with 1 × 105 red bloods cells (RBCs) infected with Plasmodium yoelii 17XNL diluted in 200 μL of saline, prepared from frozen stock. Parasitemia, the percentage of infected red blood cells, was monitored using flow cytometry beginning on day 5 p.i. and continuing every other day until clearance of the parasite using blood taken from the tails of infected mice. From each mouse, 5 μL of whole blood was diluted in 100 μL of cold PBS, fixed in 0.00625% glutaraldehyde, and then stained. The staining panel included CD45.2-APC (clone 104; Biolegend, San Diego, CA), Ter119-APC/Cy7 (clone TER-119; Biolegend, San Diego, CA), dihydroethidium (MilliporeSigma, St. Louis, MO), and Hoechst 33342 (MilliporeSigma, St. Louis, MO); samples were then resuspended in flow cytometry buffer and analyzed. Single cells were gated by Ter119+CD45.2− followed by gating the infected subpopulation on dihydroethidium+Hoechst 33342+ to identify percent parasitemia. There were 2 experiments total (4 mice/group/experiment).
Denny J.E, & Schmidt N.W. (2019). Oral Administration of Clinically Relevant Antimalarial Drugs Does Not Modify the Murine Gut Microbiota. Scientific Reports, 9, 11952.
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5

Intravenous Mouse Malaria Infection

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Donor mice were injected intravenously (IV) with thawed 105 infected red blood cells (iRBCs) with either Plasmodium yoelii 17XNL or Plasmodium berghei ANKA. Blood was collected on day 5 post infection (p.i.) via retro-orbital bleed. The blood was counted for iRBCs with Giemsa stain and total RBCs (using Hemocytometer) and diluted in 0.9% saline (Teknova, Hollister, CA) at a concentration of 105 iRBCs / 200 μl. Experimental mice were infected with 105 iRBCs IV unless indicated.
For parasitemia, approximately 5 μl whole blood was collected from tail snip in cold 100 μl 1X PBS in a 96 well plate on ice. The cells were fixed in 0.00625% glutaraldehyde, stained with conjugated antibodies, and subjected flow-cytometry analysis for evaluation of percent parasitemia. The conjugated antibodies for staining panel were CD45.2-APC clone 104; Biolegend, San Diego, CA), Ter 119-APC/Cy7 clone TER-119; Biolegend, San Diego, CA), dihydroethidium MilliporeSigma, St. Louis, MO), and Hoechst 33342 MilliporeSigma, St. Louis, MO). Forward and side scatter singlets were gated on Ter119+CD45.2− for RBC. RBCs were gated on Hoechst+dihydroethidium+ to calculate the percent of iRBC (% Parasitemia). Parasitemia was tacked every other day beginning day 5 to clearance of parasite unless indicated.
Mandal R.K., Denny J.E., Namazzi R., Opoka R.O., Datta D., John C.C, & Schmidt N.W. (2021). Dynamic modulation of spleen germinal center reactions by gut bacteria during Plasmodium infection. Cell reports, 35(6), 109094.
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6

Monitoring Plasmodium yoelii Infection in Mice

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C57BL/6N mice were injected intravenously with 1 × 105 red blood cells (RBCs) infected with Plasmodium yoelii 17XNL diluted in 200 μL of saline, passaged from donor infected mice. Parasitemia, the percentage of infected red blood cells, was monitored using flow cytometry beginning on day 5 p.i. and continuing every other day until clearance of the parasite. Blood was collected from the tails of infected mice. From each mouse, ~ 5 μL of whole blood was diluted in 100 μL of cold PBS, fixed in 0.00625% glutaraldehyde, and then stained. The staining panel included CD45.2-APC (clone 104; Biolegend, San Diego, CA), Ter119-APC/Cy7 (clone TER-119; Biolegend, San Diego, CA), dihydroethidium (MilliporeSigma, St. Louis, MO), and Hoechst 33342 (MilliporeSigma, St. Louis, MO); samples were then resuspended in flow cytometry buffer and analyzed. Infected red blood cells were identified by first gating on single cells followed by gating on Ter119+CD45.2− red blood cells. Infected red blood cells were identified as dihydroethidium+Hoechst 33342+.
Mandal R.K., Denny J.E., Waide M.L., Li Q., Bhutiani N., Anderson C.D., Baby B.V., Jala V.R., Egilmez N.K, & Schmidt N.W. (2020). Temporospatial shifts within commercial laboratory mouse gut microbiota impact experimental reproducibility. BMC Biology, 18, 83.
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7

Measuring Plasmodium Parasitemia in Mice

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Blood samples were taken through tail snips at regular intervals ranging from days 4–30 post infection. Percent parasitemia evaluated as percent infected red blood cells per total red blood cells was assessed through blood smear or flow cytometry. For blood smear evaluation, fixed and giemsa stained thin blood smears, parasitized versus total red blood cells were counted at 1000× magnification. For flow cytometry evaluation, a blood droplet was added to PBS, fixed with 0.00625% gluteraldehyde, and stained with CD45.2-APC (clone 104; Biolegend, San Diego, CA), Ter119-APC/Cy7 (clone TER-119; Biolegend, Sand Diego, CA), dihydroethidium (Sigma Aldrich, St. Louis, MO), and Hoechst 33342 (Sigma Aldrich; St. Louis, MO). Parasitized red blood cells were defined as CD45.2− Terr119+ dihydroethidium+ Hoechst+ cells.
Waide M.L., Polidoro R., Powell W.L., Denny J.E., Kos J., Tieri D.A., Watson C.T, & Schmidt N.W. (2020). Gut Microbiota Composition Modulates the Magnitude and Quality of Germinal Centers during Plasmodium Infections. Cell reports, 33(11), 108503.
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8

Measuring Plasmodium Parasitemia in Mice

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Blood samples were taken through tail snips at regular intervals ranging from days 4–30 post infection. Percent parasitemia evaluated as percent infected red blood cells per total red blood cells was assessed through blood smear or flow cytometry. For blood smear evaluation, fixed and giemsa stained thin blood smears, parasitized versus total red blood cells were counted at 1000× magnification. For flow cytometry evaluation, a blood droplet was added to PBS, fixed with 0.00625% gluteraldehyde, and stained with CD45.2-APC (clone 104; Biolegend, San Diego, CA), Ter119-APC/Cy7 (clone TER-119; Biolegend, Sand Diego, CA), dihydroethidium (Sigma Aldrich, St. Louis, MO), and Hoechst 33342 (Sigma Aldrich; St. Louis, MO). Parasitized red blood cells were defined as CD45.2− Terr119+ dihydroethidium+ Hoechst+ cells.
Waide M.L., Polidoro R., Powell W.L., Denny J.E., Kos J., Tieri D.A., Watson C.T, & Schmidt N.W. (2020). Gut Microbiota Composition Modulates the Magnitude and Quality of Germinal Centers during Plasmodium Infections. Cell reports, 33(11), 108503.
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9

Murine Malaria Parasitemia Quantification

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Donor mice were injected intravenously (IV) with thawed 10 5 infected red blood cells (iRBCs) with either Plasmodium yoelii 17XNL or Plasmodium berghei ANKA. Blood was collected on day 5 post infection (p.i.) via retro-orbital bleed. The blood was counted for iRBCs with Giemsa stain and total RBCs (using Hemocytometer) and diluted in 0.9% saline (Teknova, Hollister, CA) at a concentration of 10 5 iRBCs / 200 µl. Experimental mice were infected with 10 5 iRBCs IV unless indicated.
For parasitemia, approximately 5 µl whole blood was collected from tail snip in cold 100 µl 1X PBS in a 96 well plate on ice. The cells were fixed in 0.00625% glutaraldehyde, stained with conjugated antibodies, and subjected flow-cytometry analysis for evaluation of percent parasitemia. The conjugated antibodies for staining panel were CD45.2-APC (clone 104; Biolegend, San Diego, CA), Ter 119-APC/Cy7 (clone TER-119; Biolegend, San Diego, CA), dihydroethidium (MilliporeSigma, St. Louis, MO), and Hechst 33342 (MilliporeSigma, St. Louis, MO). Forward and side scatter singlets were gated on Ter119 + CD45.2 -for RBC. RBCs were gated on Hoechst + dihydroethidium + to calculate the percent of iRBC (% Parasitemia). Parasitemia was tacked every other day beginning day 5 to clearance of parasite unless indicated.
Mandal R.K., Denny J.E., Namazzi R., Opoka R.O., Datta D., John C.C., & Schmidt N.W. (2021). Dynamic modulation of spleen germinal center reactions by gut bacteria during Plasmodium infection.
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10

Comprehensive Tumor Immune Profiling by scRNA-seq

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Young and old mice were injected subcutaneously with 2×105 B16F10 cells per mouse. The tumors were visible 5 days after engraftment and then the volumes were measured every 2 days. Tumor volumes were determined by caliper measurement using the formula V = length×width2/2. Finally, tumor tissues were excised and weighed at indicated time point (17 days after engraftment). Next, freshly isolated tumor tissues were minced into approximately 1 mm3 cubic pieces and digested using 0.1% collagenase IV (LS004186, Worthington), 0.002% DNAse I (D8071, Solarbio), and 0.01% hyaluronidase (H3506-1G, SIGMA), then incubated on a rocker at 37°C for 40–50 min (according to tumor size). The digested cells were filtered through a 70 µm cell strainer and washed twice with cold PBS (phosphate-buffered saline) added 2% FBS (Fetal bovine serum) buffer. The remaining cells were stained with APC-CD45.2 (Clone 104; BioLegend) and 7AAD (Part 76332; Lot B226294 Biolegend) for 30 min at 4°C, then washed and suspended in PBS (2% FBS) buffer for flow cytometric sorting using FACS Aria II Cell Sorter (BD Biosciences). Sorted CD45.2+7AAD- cells with a purity greater than 95% and viability higher than 90% were used for 10X genomics scRNA-seq.
Zhang C., Lei L., Yang X., Ma K., Zheng H., Su Y., Jiao A., Wang X., Liu H., Zou Y., Shi L., Zhou X., Sun C., Hou Y., Xiao Z., Zhang L, & Zhang B. (2021). Single-cell sequencing reveals antitumor characteristics of intratumoral immune cells in old mice. Journal for Immunotherapy of Cancer, 9(10), e002809.
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