Red blood cells were lysed with RBC lysis buffer (Invitrogen) and isolated splenocytes were stained with Live/Dead Aqua (ThermoFisher), or Fixable Viability Dye eF780 (ThermoFisher) for 20 min at 4°C in the absence of FBS, to differentiate live and dead cells. Cells were subsequently stained for surface markers (Supplemental table 1 ) in the presence of Fc block (αCD16/32, BioLegend) and 2% FBS. Cells expressing NP-specific BCRs were detected by staining with NP-PE (BioSearch). HEL peptide-loaded MHC II was detected using a mAb clone C3H4 (16 (link)) (kindly provided by Dr. Ron Germain, NIAID, Bethesda, MD), which was biotinylated and detected using streptavidin-PE-TexasRed (BD). After washing, cells were fixed in 1% paraformaldehyde (PFA) and stored at 4°C until analysis. Cells were analyzed on an LSRII flow cytometer (BD) and data were analyzed using FlowJo (v.10, Tree Star). See Figure 1A for gating strategies.
Fc block α cd16 32
Manufactured by BioLegend
Fc block α-CD16/32 is a monoclonal antibody that binds to the Fc receptors CD16 and CD32. It is commonly used in flow cytometry and cell-based assays to block non-specific Fc receptor-mediated binding, which can improve the specificity and sensitivity of the analysis.
Automatically generated - may contain errors
Lab products found in correlation
Cd11b microbead kit, by Miltenyi Biotec (2 mentions)
Facsaria 2 sorp, by BD (2 mentions)
Pe cy7 α cd19 1d3, by BD (2 mentions)
Fitc α cd11b m1 70, by BD (2 mentions)
Bv510 α f4 80 bm8, by BioLegend (2 mentions)
Bv711 α siglec f e50 2440, by BD (2 mentions)
Violetfluor 450 α ly 6g gr1 rb6 8b5, by Cytek Biosciences (2 mentions)
Alexafluor 647 α cd102 3c4 mic2 4, by BioLegend (2 mentions)
Ls column, by Miltenyi Biotec (2 mentions)
Streptavidin pe texas red, by BD (1 mentions)
Fixable viability dye ef780, by Thermo Fisher Scientific (1 mentions)
Rbc lysis buffer, by Thermo Fisher Scientific (1 mentions)
Live dead aqua, by Thermo Fisher Scientific (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
3 protocols using fc block α cd16 32
1
Multiparameter Flow Cytometry Analysis
2
Isolation and Characterization of Murine Myeloid Cells
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Three to four mice were pooled for each sample. PECs were collected and washed. Cells were counted and CD11b+ cells were selected by MACS column purification using the CD11b MicroBead kit and LS columns (Miltenyi Biotec). CD11b+ cells were stained with Fc block α-CD16/32 (Biolegend) and then PE-Cy7-α-CD19 (1D3, Tonbo), Fitc-α-CD11b (M1/70, BD Biosciences), BV510-α-F4/80 (BM8, Biolegend), BV711-α-Siglec F (E50-2440, BD Biosciences), Violetfluor 450-α-Ly-6G (GR1) (RB6-8B5, Tonbo), and Alexafluor 647-α-CD102 (3C4 (MIC2/4), Biolegend). Samples were run on a BD FACS Aria II SORP and collected in D10 media (for LD-PCR). Afterward, the samples were counted and frozen for LD-PCR (in media).
3
Isolation and Characterization of Murine Myeloid Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Three to four mice were pooled for each sample. PECs were collected and washed. Cells were counted and CD11b+ cells were selected by MACS column purification using the CD11b MicroBead kit and LS columns (Miltenyi Biotec). CD11b+ cells were stained with Fc block α-CD16/32 (Biolegend) and then PE-Cy7-α-CD19 (1D3, Tonbo), Fitc-α-CD11b (M1/70, BD Biosciences), BV510-α-F4/80 (BM8, Biolegend), BV711-α-Siglec F (E50-2440, BD Biosciences), Violetfluor 450-α-Ly-6G (GR1) (RB6-8B5, Tonbo), and Alexafluor 647-α-CD102 (3C4 (MIC2/4), Biolegend). Samples were run on a BD FACS Aria II SORP and collected in D10 media (for LD-PCR). Afterward, the samples were counted and frozen for LD-PCR (in media).
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