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MACS Cell Pre-Enrichmentfor flow sorting and analysisSaves flow sorting timeImproves cell viabilityEnhances quality offlow cytometry data

MACS Cell Pre-Enrichment for flow sortingTime-saving cell processing, improved sort qualitySaving flow sorting timeby MACS Cell Pre-EnrichmentImproving cell viabilityand sort quality Total sorting time reduced by up to 80% Removal of cell debris prior to cell sorting Target population can be sorted directlyafter pre-enrichment Reduction of sorting time increases cell viability rateSorting large numbers of cells or rare cell populationscan take a long time and often compromises cell viability.Pre-enrichment of these cells based on MACS MicroBeadTechnology solves both of these problems as it takes lessthan 30 min and eliminates all unwanted cells and debris.For example, magnetic separation of 5 10⁸ cells prior toprocessing on high-speed cell sorters effectively reducesthe sorting time from 7 hours to 1.4 hours. MACSMicroBeads are compatible with flow sorting and cells canbe sorted and analyzed right after pre-enrichment withoutfurther treatment.MACS Cell Pre-Enrichment saves sorting time by eliminatingcell debris and non-target cells. Removing cell debris beforesorting increases the efficiency and minimizes the percentageof abort events caused by non-specific influence of thedebris. Using a clean sample is important for precise highspeed cell sorting, in particular for sorting rare cell populations( 1%) where high abort event rates often lead to a low cellyield. Shorter total sorting time also increases the percentageof viable cells since the cells spend less time in the collectiontubes before and after processing.PBMCsPre-enriched sample12.52.5811.12.2730.810014.22.810³10²4 5 6 7 8 9 10 11 12 13 14 15Time spent on cell sorter (h)Without pre-enrichment10⁵10⁴10¹With CD4 cell pre-enrichmentFigure 1: Time spent for sorting of CD4 subsets with and withoutpre-enrichment. The bar graph demonstrates an example of total timespent on a cell sorter when sorting mouse CD4 cell subpopulationsdirectly (light blue bar) or pre-enriched by MACS Technology (dark bluebar) at a sorting speed of 20,000 events/s. Depending on cell numbers,time savings can be up to five-fold, from 14 h to less than 3 h whensorting 1 10⁹ cells.10³10²10¹0350 100 150 200 250Deadcells10⁴1250Forward scatter10³10⁴10⁵010³10⁴10⁵Propidium iodidePropidium iodideDirectsortingSorting 5.6CD425015010050 100 150 200 250CD41.1100200Forward scatter6.91.441508.31.75Debris2009.71.96Side scatter9Input cell number ( 10⁸)13.92.8250CD410Side ropidium iodide010³10⁴10⁵Propidium iodideFigure 2: Higher cell purity and viability with pre-enrichment.CD4 subsets were sorted from human PBMCs with and without preenrichment. Due to gentle pre-enrichment by the CD4 T Cell IsolationKit and shorter subsequent sorting, the number of viable cells is muchhigher (93.5%) than in the sample sorted without pre-enrichment (78.6%).2

MACS Cell Pre-Enrichment for flow sortingSelected productsSelected cell separation reagents for pre-enrichment of human cellsPre-enrichmentmarkersFlow sorting subsets(examples)Reagents for positiveselection of desired cellsReagents for depletionof unwanted cellsCD45T cells: CD45 CD3 CD19–B cells: CD45 CD19 CD3–CD45 MicroBeads130-045-801CD3T helper cells: CD3 CD4 CD8 –Cytotoxic T cells: CD3 CD8 CD4 –CD3 MicroBeads130-050-101Pan T Cell Isolation Kit130-096-535CD4Naive T cells: CD4 CD45RA CCR7 CD62L–CD45RO–Effector memory T cells: CD4 CD45RO CCR7–CD62L–Central memory T cells: CD4 CD45RO CD45RA –CCR7–CD62L Regulatory T cells: CD4 CD25 CD127loCD4 MicroBeads130-045-101CD4 T Cell Isolation Kit130-096-533CD8Naive T cells: CD8 CD45RA CCR7 CD62L–CD45RO–Effector memory T cells: CD8 CD45RO CCR7-CD62L–Central memory T cells: CD8 CD45RO CD45RA –CCR7 –CD62L CD8 MicroBeads130-045-201CD8 T Cell Isolation Kit130-096-495CD19Naive B cells: CD19 IgD CD27 –Memory B cells: CD19 CD27 Ig D/A CD19 MicroBeads130-050-301REAlease CD19MicroBead Kit130-117-034Pan B Cell Isolation Kit130-101-638CD11bM-MDSC: CD11b CD15–CD14 CD33 HLADRloG-MDSC: CD11b CD15 CD56b CD11b MicroBeads130-049-601CD34HSC: CD34 CD38 –Thy–1 CD45RA – Flt3 CD7–CD10 –CD34 MicroBead Kit UltraPure130-100-453Lineage Cell Depletion Kit130-092-211Reagents for depletionof unwanted cellsSelected cell separation reagents for pre-enrichment of mouse cellsPre-enrichmentmarkersFlow sorting subsets(examples)Reagents for positiveselection of desired cellsCD45T cells: CD45 CD3 CD19–B cells: CD45 CD19 CD3–CD45 MicroBeads130-052-301CD3T helper cells: CD3 CD4 CD8 –Cytotoxic T cells: CD3 CD8 CD4 –CD3ε MicroBead Kit130-094-973Pan T Cell Isolation Kit130-096-535CD4Naive T cells: CD4 CD44loCD62L Regulatory T cells: CD4 CD25 Foxp3 *CD4 (L3T4) MicroBeads130-117-043CD4 T Cell Isolation Kit130-104-454CD8Naive T cells: CD8 CD44loCD62L CD8a (Ly-2) MicroBeads130-117-044CD8 T Cell Isolation Kit130-104-075CD19B220 (CD45RA)Naive B cells: CD19/B220 IgM IgD CD23 Follicular B cells: CD19/B220 IgMloIgDhiCD21intCD23 Marginal B cells: CD19/B220 IgM IgDloCD1dhiCD21hiCD23–CD19 MicroBeads130-052-201CD45R (B220) MicroBeads130-049-501Pan B Cell Isolation Kit II130-104-443CD11cCD11b cDC: CD11c MHCII CD11bhiCD172a XCR1 cDC: CD11c MHCII CD103 XCR1 CD11c MicroBeadsUltraPure130-108-338Pan DC Isolation Kit130-100-875LineagedepletionHSC: Lin–Sca-1 c-kit CD48 –CD150 Direct Lineage CellDepletion Kit130-110-470* FoxP3 is an intracellular marker. Cells from FoxP3 reporter mice are suitable for flow sorting.3

MACS Cell Pre-Enrichment for flow analysisEnhanced quality of flow cytometry dataImproving the analysisof rare cell populationsAnalyzing rare populations such as tumor-infiltratingleukocytes or lymphocytes (TILs) with cell analyzers is oftenchallenging, as these small target populations can easily belost in the background noise. Obtaining a number of eventsthat is large enough for a detailed and significant analysisof subpopulations can be very time consuming, especiallywhen analyzing multiple samples.AMACS Technology enables pre-enrichment of cells prior toanalysis in quick and easy steps. Pre-enriched samples canthen be directly analyzed by flow cytometry in a shortertime and a more detailed manner, which ultimately resultsin an increased quality of flow cytometry data.With pre-enrichmentBWithout pre-enrichmentPre-enriched CD45 TILsCD45 leukocytesin bulk tumor96.97 %Side scatterSide scatter2.27 %CD19-APCGated onCD45 CD3ε cellsGated onCD45 CD3ε CD11b– 11b cDCs38.10%Inflammatory DC s42.86%Anti-Ly-6C-PE-Vio 770Anti-Ly-6C-PE-Vio 770Gated on CD45 CD11b CD11c Ly6G – cellsGated on CD45 CD11b CD11c Ly6G – cellsCD8 T cells28.46%CD8a-APCGated onCD45 CD3ε -Ly-6C-PE-Vio 770Gated on CD45 CD11b CD11c Ly6G – cellsCD11c 19.76%B cells16.72%CD19-APCGated onCD45 CD3ε– CD11b– cellsDCsCD8a-APCDN CD3ε cells24.72%Anti-MHC Class II-APCB cells1.82%CD4 Tcells44.95%T cellsCD4-APC-Vio 770CD11c 2.23%TAMAnti-MHC Class II-APCB cellsCD8 T cells16.49%CD11c-APC-Vio 770DN CD3ε cells49.48%Anti-MHC Class II-APCCD4-APC-Vio 770T cellsTAMAnti-MHC Class II-APCCD4 T cells31.96%B cellsCD11c-APC-Vio 770CD45-VioGreenCD45-VioGreen CD11b cDCs InflammatoryDC s 36.71%51.26%Anti-Ly-6C-PE-Vio 770Gated on D45 CD11b CD11c Ly6G – cellsFigure 3: Improving analysis of tumor-infiltrating leukocytes (TILs) using MACS Cell Pre-Enrichment. Total (A) or pre-enriched CD45 TILs (B)from B16-F10 tumors were labeled with the fluorochrome-conjugated REAfinity Recombinant Antibodies and analyzed by flow cytometry usingthe MACSQuant Analyzer 10. Gated populations show viable cells. Compared to the samples analyzed without pre-enrichment, cell numbers ofthe desired subpopulations were significantly increased after MACS Cell Pre-Enrichment. DC: dendritic cells, TAM: tumor-associated macrophages4

MACS Cell Pre-Enrichment for flow analysisGetting reliable flow cytometry data fasterCell pre-enrichmentspeeds up cell analysisthe time to get significant flow cytometry data. Particularlywhen analyzing large numbers of samples the total analysistime can be reduced considerably.It takes quite some time to acquire enough cells for theaccurate flow cytometry analysis of a rare subpopulation.Enrichment of the cells prior to analysis however minimizesWithout pre-enrichment0.53%10¹10-1-1 0 110¹10²10³10³Lag3-FITC10²CD223-VioBright 0²127.8%0 15.5%-1-1 0 223-VioBright 515CD4-PE-VioCD4-PE-Vio61561591.6%10²Acquisition rate: 2,000 event/sRequiredanalysis time: 8 min/sample 5,000 target events (CD8 ) 940,000 total eventsConditions:10³10¹ Events needed:With pre-enrichment10-1-1 0 1Percentage of CD8 cellsamong living lymphocytes: 0.53%10¹CD8β-VioBlueCD8β-VioBlue10² 55.7%0.47% Percentage of CD8 cellsamong living lymphocytes: 91.6% Acquisition rate: 2,000 event/s61.6%10²Requiredanalysis time:10¹10 15.4%22.5%-1-1 0 lue 5 s/sampleEvents needed: 5,000 target events (CD8 ) 5,500 total eventsFigure 4: Example of time calculation for flow cytometry analysis of TILs. Cells derived from B16-F10 tumors were analyzed by flow cytometry with orwithout pre-enrichment using CD8 (TIL) MicroBeads. Cells were stained with REAfinity Antibodies CD4-PE-Vio 615, CD8β-VioBlue , CD223-VioBright 515,and PD1-APC. Theoretical acquisition time is calculated based on achieving 5,000 target events.Estimated time required to acquire specified TIL populationsTarget populationCD45 cellsCD4 T cellsCD8 T cellsPan T cellsTumor riched*BulkPre-enriched*Target tal collected events5 10⁵2 10⁴7.9 10⁶5.4 10⁴2.8 10⁶4.4 10⁴8.1 10⁵3.2 10⁴Acquisition time/sample**4 min0.2 min66 min0.5 min23 min0.4 min7 min0.3 minTotal acquisition time for12 samples*** 1 h 10 min 10 h 11 min 3.5 h 10 min 1 h 10 min*Isolation using CD45 (TIL) MicroBeads, CD8 (TIL) MicroBeads, CD4 (TIL) MicroBeads, or CD4/CD8 (TIL) MicroBeads, respectively.** Acquisition rate: 2,000 events/s on MACSQuant Analyzer 10. *** Includes 45 s automated mixing and rinsing between samples.Selected products for TIL pre-enrichment CD45 (TIL) MicroBeads, human (# 130-118-780) CD45 (TIL) MicroBeads, mouse (# 130-110-618) CD4/CD8 (TIL) MicroBeads, mouse (# 130-116-480)CD4 (TIL) MicroBeads, mouse (# 130-116-475)CD8 (TIL) MicroBeads, mouse (# 130-116-478)5