8. Supplies: scissors, tweezers, tweezers (elbow), beaker, pipette,
tips, foil paper, masks, gloves, ice-old metal pieces, singleedged razor blades, CellTrics® disposable filters, 30 μm porosity (Partec, Mu ¨nster, Germany), petri dish, glass slide, 2-mL
protein LoBind tubes (Eppendorf), 10-μL/20-μL/200-μL/
1000-μL tips (epT.I.P.S.
® LoRetention Reloads).
9. Instruments: MoFlo/BD sorter, phase contrast microscope,
LUNA-FL™ dual fluorescence cell counter.
2.4 Extraction and
Amplification of RNA
from Protoplasts and
Nuclei
1. Lysis Buffer (sufficient for 20 samples):
μL
TaKaRa 10Â lysis buffer
20
SUPERase RNase inhibitor
90
RNasin
® ribonuclease inhibitors
90
H 2 O to a total volume of 180 μL
2. RNase inhibitors: 0.5 U/μL SUPERase In™ RNase Inhibitor
(ThermoFisher catalog number: AM2694) and 0.5 U/μL
RNasin(R) RNase Inhibitor (Promega catalog number:
N2111).
3. RNAse-free microcentrifuge tubes, 0.6 and 1.5 mL. RNAsefree 96-well plates.
2.5 Flow Cytometry
Standards
1. Standard fluorescent microspheres (10 μm Flow-CheckTM
Fluorospheres (Beckman Coulter, Brea, CA, or similar).
2. Pollen: Standard biological particles from Polysciences (Warrington, PA) include:
Item
Binomial
Diameter
(μm)
Catalog
number
Paper Mulberry
Pollen
Morus papyrifera L.
12–13
07670-1
Common
Ragweed
Pollen
Ambrosia artemisiifolia L. 19–20
07673-1
Club Moss
Spores
a
Lycopodium L. and
Diphasiastrum Holub
28
16,867-1
Pecan Pollen
Carya illinoinensis
(Wangenh.) K.Koch
45–52
07671-1
Corn Pollen
Zea mays L.
85–90
07664-1
a
Preferred source species are Lycopodium clavatum (wolf’s-foot clubmoss) and
Diphasiastrum digitatum (common groundcedar)
272
David W. Galbraith and Guiling Sun
tips, foil paper, masks, gloves, ice-old metal pieces, singleedged razor blades, CellTrics® disposable filters, 30 μm porosity (Partec, Mu ¨nster, Germany), petri dish, glass slide, 2-mL
protein LoBind tubes (Eppendorf), 10-μL/20-μL/200-μL/
1000-μL tips (epT.I.P.S.
® LoRetention Reloads).
9. Instruments: MoFlo/BD sorter, phase contrast microscope,
LUNA-FL™ dual fluorescence cell counter.
2.4 Extraction and
Amplification of RNA
from Protoplasts and
Nuclei
1. Lysis Buffer (sufficient for 20 samples):
μL
TaKaRa 10Â lysis buffer
20
SUPERase RNase inhibitor
90
RNasin
® ribonuclease inhibitors
90
H 2 O to a total volume of 180 μL
2. RNase inhibitors: 0.5 U/μL SUPERase In™ RNase Inhibitor
(ThermoFisher catalog number: AM2694) and 0.5 U/μL
RNasin(R) RNase Inhibitor (Promega catalog number:
N2111).
3. RNAse-free microcentrifuge tubes, 0.6 and 1.5 mL. RNAsefree 96-well plates.
2.5 Flow Cytometry
Standards
1. Standard fluorescent microspheres (10 μm Flow-CheckTM
Fluorospheres (Beckman Coulter, Brea, CA, or similar).
2. Pollen: Standard biological particles from Polysciences (Warrington, PA) include:
Item
Binomial
Diameter
(μm)
Catalog
number
Paper Mulberry
Pollen
Morus papyrifera L.
12–13
07670-1
Common
Ragweed
Pollen
Ambrosia artemisiifolia L. 19–20
07673-1
Club Moss
Spores
a
Lycopodium L. and
Diphasiastrum Holub
28
16,867-1
Pecan Pollen
Carya illinoinensis
(Wangenh.) K.Koch
45–52
07671-1
Corn Pollen
Zea mays L.
85–90
07664-1
a
Preferred source species are Lycopodium clavatum (wolf’s-foot clubmoss) and
Diphasiastrum digitatum (common groundcedar)
272
David W. Galbraith and Guiling Sun