To perform surface staining, 1 × 106 cells were placed in individual wells of a 96-well round bottom plate and incubated with the appropriate antibody cocktails for 15 min at 4°C on a slow rocker. After the staining, cells were fixed in a solution of 2% ultrapure formaldehyde (Polysciences, Inc., Warrington, PA, USA) in FACS buffer for 20 min on ice, washed twice, and analyzed the following day on the Canto II (BD Biosciences) or FACSCalibur (BD Biosciences). Intracellular staining was performed using Cytofix/Cytoperm Fixation/Permeabilization Solution Kit with BD GolgiStop (BD biosciences) according to the manufacturer’s instruction. Flow cytometry acquisition was performed on an LSRIISorp. Data were analyzed using FACS Express or FlowJo software (Tree Star, Inc., Ashland, OR, USA). Antibodies against CD45 (clone 30-F11, BD Pharmingen), CD3 (clone 145-2C11, BD Pharmingen), CD4 (clone GK1.5, BD Pharmingen), CD8 (clone 5H10, Biolegend), T-bet (clone eBio4B10, eBioscience), IL-17A (clone ebio17B7, eBioscience), IL-4 (clone B11B, Biolegend), IFNγ (clone XMG 1.2, eBioscience), IL-22 (clone A3.6M, eBioscience and clone poly5164, Biolegend), TGF-β (clone 11A5, Biolegend), IL-17F (clone ebio18F10, eBioscience), NKP46 (clone 29A 1.4, Biolegend), c-Kit (clone 2B8, Biolegend), Sca-1 (clone D7, BD Pharmingen), and CD127 (clone A7R34, eBioscience) were used.
Cd45 clone 30 f11
Manufactured by BD
Sourced in United States
CD45 (clone 30-F11) is a mouse monoclonal antibody that recognizes the CD45 antigen, also known as the Leukocyte Common Antigen. CD45 is a transmembrane protein tyrosine phosphatase that is expressed on the surface of all nucleated hematopoietic cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd11b clone m1 70, by BD (4 mentions)
Ly6g clone 1a8, by BD (4 mentions)
Facscalibur, by BD (3 mentions)
Ifn γ clone xmg1, by Thermo Fisher Scientific (2 mentions)
Cd105 clone mj7 18, by BioLegend (2 mentions)
Siglecf clone e50 2440, by BD (2 mentions)
Ly6g clone 1a8, by BioLegend (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Collagenase d, by Merck Group (2 mentions)
Anti mouse cd3 clone 17a 2, by BD (2 mentions)
F4 80 clone bm8, by Thermo Fisher Scientific (2 mentions)
Cd11b clone m1 70, by Thermo Fisher Scientific (2 mentions)
Facscanto 2, by BD (2 mentions)
Cd4 clone rm4 5, by BioLegend (2 mentions)
Canto 2, by BD (1 mentions)
Cd127 clone a7r34, by Thermo Fisher Scientific (1 mentions)
Ultrapure formaldehyde, by Polysciences (1 mentions)
Cd3 clone 145 2c11, by BD (1 mentions)
Cd4 clone gk1, by BD (1 mentions)
T bet clone ebio4b10, by Thermo Fisher Scientific (1 mentions)
22 protocols using cd45 clone 30 f11
1
Multiparametric Flow Cytometry Analysis
2
Immunohistochemical Analysis of Lacrimal Gland
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Lacrimal glands were surgically excised and immersed in 4% paraformaldehyde overnight at 4°C. The tissue blocks were washed, dehydrated, embedded in paraffin, cut to a thickness of 3 mm. The cells were counted that stained positively for CD4 (clone H129.9, 10μg/mL; BD Bioscience, San Diego, CA), CD8α (clone 53e6.7, 3.125μg/mL; BD Bioscience), CD11b (clone M1/70, 6.25 μg/mL; BD Bioscience),CD45 (clone 30-F11, 10 μg/mL; BD Bioscience), CD103 (clone 2E7, 10 μg/mL; Biolegend, San Diego, CA), paraffin sections were stained with the abovementioned primary antibodies and appropriate biotinylated secondary antibodies (all from BD Pharmingen, San Diego, CA) using a staining kit ( Vectastain Elite ABC kit; Vector, Burlingame, CA) and reagents (Nova-Red; Vector). Secondary antibody alone and appropriate anti-mouse isotype (BD Biosciences) controls were also performed. Two sections from each animal were examined and photographed with a microscope (Imager.Z1; Carl Zeiss Meditec, Oberkochen, Germany). Positively stained cells were counted in the stroma of the LG using image-analysis software (NIS Elements Software, version 3.0 BR; Nikon). Results were expressed as the number of positive cells per mm2.
3
Immunophenotypic Analysis of Mesenchymal Stem Cells
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For the immunophenotypic analysis, third passage MSCc and MSCd were dissociated
and suspended in blocking solution containing cold PBS supplemented with 0.5%
bovine serum albumin (BSA). The cells were treated with rat Fc block (CD32,
Cat#550271; BD Biosciences, San Jose, CA, USA) for 20 minutes before incubation
with antibodies. The following antibodies conjugated with fluorescein
isothiocyanate (FITC), phycoerythrin (PE), or biotin were used: CD29 (clone
Ha2/5, Cat#555005, dilution 1:50; BD Biosciences), CD90-1 (clone OX-7,
Cat#551401, dilution 1:25; BD Biosciences), CD45 (clone 30-F11, Cat#553077,
dilution 1:50; BD Biosciences), and CD34 (clone RAM34, Cat#551387, dilution
1:50; BD Biosciences). The corresponding isotypes (BD Biosciences) were used as
nonspecific binding controls. After incubation at 4ºC for 20 minutes, the cells
were washed with PBS/0.5% BSA, centrifuged at 300 xg for 5 minutes, and
suspended in PBS for data acquisition. DAPI 0.1 µg/mL (Cat# D9452,
Sigma-Aldrich) was added to exclude dead cells. The samples were acquired in a
BD FACSAria II flow cytometer (BD Biosciences) and the resulting data were
analyzed using the software FlowJo, version 10.1.
and suspended in blocking solution containing cold PBS supplemented with 0.5%
bovine serum albumin (BSA). The cells were treated with rat Fc block (CD32,
Cat#550271; BD Biosciences, San Jose, CA, USA) for 20 minutes before incubation
with antibodies. The following antibodies conjugated with fluorescein
isothiocyanate (FITC), phycoerythrin (PE), or biotin were used: CD29 (clone
Ha2/5, Cat#555005, dilution 1:50; BD Biosciences), CD90-1 (clone OX-7,
Cat#551401, dilution 1:25; BD Biosciences), CD45 (clone 30-F11, Cat#553077,
dilution 1:50; BD Biosciences), and CD34 (clone RAM34, Cat#551387, dilution
1:50; BD Biosciences). The corresponding isotypes (BD Biosciences) were used as
nonspecific binding controls. After incubation at 4ºC for 20 minutes, the cells
were washed with PBS/0.5% BSA, centrifuged at 300 xg for 5 minutes, and
suspended in PBS for data acquisition. DAPI 0.1 µg/mL (Cat# D9452,
Sigma-Aldrich) was added to exclude dead cells. The samples were acquired in a
BD FACSAria II flow cytometer (BD Biosciences) and the resulting data were
analyzed using the software FlowJo, version 10.1.
4
Multiparametric Flow Cytometry Analysis
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The following antibodies were used: CD45 (clone 30-F11), CD11b (M1/70), KLRG1 (2F1), CD103 (M290), NKg2a (20d5), Ly6C (AL-21), Ly6G (1A8), PD-L1 (MIH5), I-A/I-E (M5/114), CD11c (HL3), PDCA1 (927), CD64 (X54-5/7.1), B220 (RA3-6B2), CD24 (M1/69), CD4 (GK1.5), CD25 (3C7), CD3 (500A2), NKp46 (29A1.4), TNF-α (MP6-XT22), IFN-γ (XMG1.2), H2-Kb (AF6-88.5), and H2-Db (28–14–8) were from BD Biosciences; Tim3 (RMT3-23), PD-1 (29F.1A12), CD38 (90), Gr-1 (RB6-8C5), CD206 (C068C2), CD68 (FA-11) were from BioLegend; FoxP3 (FJK-16s), T-bet (4B10), GATA-3 (TWAJ), and RORγt (AFKJS-9) were from ThermoFisher Scientific; Granzyme B (REA226) was from Miltenyi; CD8 (KT15) was from MBL. Dead cells were stained with LIVE/DEAD Yellow or Aqua fluorescent reactive dye (Life Technologies) and excluded from analyses. Murine MHC-peptide multimers were from Immudex (Copenhagen, Denmark). Cells were analyzed using an Attune NxT flow cytometer (ThermoFisher Scientific), and results were analyzed with Kaluza (Beckman Coulter) software.
5
Immune Cell Profiling in Murine Super-Infection
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Whole blood was collected from mice on days −1, 3, 7 and 9 post‐super‐infection. Blood was collected in 0.5 M EDTA, then lysed using ACK Lysis buffer, washed and resuspended in flow cytometry buffer (2% FCS + 0.5 mM EDTA in PBS). Single‐cell suspensions were blocked with anti‐CD16/32 antibody and stained with fluorochrome‐conjugated antibodies against mouse CD3 (clone 17A2; Biolegend, San Diego, CA, USA), CD4 (clone GK1.5, Biolegend), CD8 (clone 53‐6.2, BD Biosciences, Franklin Lakes, NJ, USA), CD45 (clone 30‐F11, BD Biosciences), CD11b (clone M1/70, BD Biosciences), IA/IE (M5/114; eBioscience, San Diego, CA, USA), Ly6G (clone 1A8, BD Biosciences), CD62L (clone MEL‐14, BD Biosciences) and CD69 (clone H1.2F3, BD Biosciences), and dead cells were excluded using LIVE/DEAD Near‐Infrared viability dye (#L110909, Thermo Fisher Scientific). Sphero™ Blank Calibration Particles (BD Biosciences) were added to the samples and used as reference counting beads. Cells were acquired on a BD LSR II Fortessa Cell Analyser, and data were analysed using FlowJo software (v10.6; Treestar, Inc.).
6
Tumor Immunohistochemistry in Aged Mice
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Mice were aged for 600 days and killed when visible tumor burden was apparent or at the end of the study period (day 601).28 (link) Immunohistochemisty analyses were performed on tumor sections to confirm histopathological examinations and further sub-typing of tumors was carried out using the following primary antibodies: CD3 (ab5690, Abcam), CD10 (ab951, Abcam), CD34 (ab8158, Abcam), CD45R (clone RA3-6B2, BD Biosciences, Franklin Lakes, NJ, USA), CD45 (clone 30-F11, BD Biosciences), and CD138 (ab34164, Abcam).
7
Distinguishing Intravascular and Interstitial Neutrophils
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After mice were euthanized, BALF was collected and centrifuged, and neutrophils were counted using the Kimura stain.
Intravascular and interstitial neutrophils in the lungs were distinguished by a flow cytometry-based method as previously described [19 (link)]. Briefly, an Alexa 633-labeled GR-1 antibody (clone RB6-8C5, staining kit: Invitrogen Corp., Carlsbad, CA, USA) was injected i.v. 5 min before euthanasia, labeling only intravascular neutrophils. After performing BAL, the inferior vena cava was dissected and non-adherent neutrophils were removed from the pulmonary vasculature by flushing 10 ml of PBS at 25 ml H2O through the spontaneously beating right ventricle. Lungs were removed, minced, and digested with enzyme cocktail at 37°C for 60 min. A cell suspension was prepared by passing the digested lungs through a 70 mm cell strainer (BD Falcon, Bedford, MA, USA) which lysed the erythrocytes, and the remaining leukocytes were counted. The fraction of neutrophils in the suspension was determined by flow cytometry using a FACSCalibur (Becton Dickinson, San Jose, CA, USA). Neutrophils were identified by their typical expression of CD45 (clone 30-F11, BD Biosciences-Pharmingen, San Diego, CA, USA) and GR-1 (clone RB6-8C5). The i.v. injected labeled GR-1 Ab differentiated intravascular and interstitial neutrophils.
Intravascular and interstitial neutrophils in the lungs were distinguished by a flow cytometry-based method as previously described [19 (link)]. Briefly, an Alexa 633-labeled GR-1 antibody (clone RB6-8C5, staining kit: Invitrogen Corp., Carlsbad, CA, USA) was injected i.v. 5 min before euthanasia, labeling only intravascular neutrophils. After performing BAL, the inferior vena cava was dissected and non-adherent neutrophils were removed from the pulmonary vasculature by flushing 10 ml of PBS at 25 ml H2O through the spontaneously beating right ventricle. Lungs were removed, minced, and digested with enzyme cocktail at 37°C for 60 min. A cell suspension was prepared by passing the digested lungs through a 70 mm cell strainer (BD Falcon, Bedford, MA, USA) which lysed the erythrocytes, and the remaining leukocytes were counted. The fraction of neutrophils in the suspension was determined by flow cytometry using a FACSCalibur (Becton Dickinson, San Jose, CA, USA). Neutrophils were identified by their typical expression of CD45 (clone 30-F11, BD Biosciences-Pharmingen, San Diego, CA, USA) and GR-1 (clone RB6-8C5). The i.v. injected labeled GR-1 Ab differentiated intravascular and interstitial neutrophils.
8
Characterization of Brain Immune Cells
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BILs were incubated with 0.5 μg Fc block (anti-FcyRIII/II mAb) prepared from the supernatant of 2.4G2 hybridoma cells for 30 min on ice, followed by staining with CD45 (clone 30-F11, BD Biosciences), CD11b (clone M1/70, BD Biosciences), Ly6G/C (clone RB6-8C5, BD Biosciences), or Ly6G (clone 1A8, BD Biosciences). All antibodies were added to blocked wells at 1:200, incubated for 30 min, and washed three times prior to flow cytometric analysis. Brain-infiltrating cells were gated on CD45 expression. All CD45mid/hi cells were further assessed for expression of CD11b, Ly-6C/G (Gr1), and Ly-6G (1A8). We defined inflammatory monocytes as the CD45hiCD11b+Gr1++1A8− population, neutrophils as CD45hiCD11b++Gr1+1A8+ cells, and microglia as CD45midCD11bmidGr1− cells. For phenotyping experiments involving reporter LysM:eGFP mice, we defined inflammatory monocytes as GFPmid cells, neutrophils as GFPhi cells, and microglia as GFPneg cells within a specific forward- and side-scatter gate. Flow cytometric analysis was performed on an Accuri C6 flow cytometer with sampler arm (BD Biosciences, Mountain View, CA). Files were analyzed offline using FlowJo 10.08 (Windows version; FlowJo LLC, Ashland, OR).
9
Isolation and Characterization of Islet Cells
10
Flow Cytometric Sorting of Murine Myeloid Cells
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