A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

Podsumowanie

A standard Western blotting protocol was optimized for analyzing as few as 500 hematopoietic stem or progenitor cells. Optimization involves careful handling of the cell sample, limiting transfers between tubes, and directly lysing the cells in Laemmli sample buffer.

Streszczenie

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western blotting protocol was optimized and suitable for the analysis of small numbers of HSCs (500 - 15,000 cells). Phenotypic HSCs were purified, accurately counted, and directly lysed in Laemmli sample buffer. Lysates containing equal numbers of cells were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the blot was prepared and processed following standard Western blotting protocols. Using this protocol, 2,000 - 5,000 HSCs can be routinely analyzed, and in some cases data can be obtained from as few as 500 cells, compared to the 20,000 to 40,000 cells reported in most publications. This protocol should be generally applicable to other hematopoietic cells, and enables the routine analysis of small numbers of cells using standard laboratory procedures.

Wprowadzenie

Hematopoietic stem cells (HSCs) are self-renewing cells that can give rise to all blood lineages. They are relatively rare cells in the bone marrow, rendering biochemical analyses difficult. Approaches suitable for analyzing rare cells, such as flow cytometry, have been extremely useful for quantifying relative amounts of cell surface markers and intracellular proteins. However, the analysis of intracellular proteins necessitates the use of cell permeabilization procedures to enable antibody access, and not all cell surface epitopes survive these procedures^1,2. In addition, antibodies that discriminate between different protein isoforms or cleavage products are not often available for flow cytometry, and therefore investigators still rely on Western blots for certain types of analyses.

Western blot analysis of cell lysates is a routine procedure in most laboratories. Cells can be purified under native conditions that preserve the epitopes of cell surface molecules, and cell lysates can subsequently be prepared and analyzed. However, the analysis of proteins in rare primary cell populations by Western blot can require euthanizing large numbers of animals to obtain enough cells. By making small adjustments to several steps, a conventional Western blotting protocol was able to detect proteins in relatively small numbers of HSCs (500 - 15,000, depending on the protein of interest). The adjustments include accurately counting the cells, carefully handling the cell pellet, reducing transfers of cells between tubes to minimize cell loss, and lysing a defined number of cells with a concentrated loading buffer containing proteasome and phosphatase inhibitors. Many published reports include Western blots obtained with 20,000 or more HSCs^3,4,5,6,7; this simple procedure will reduce the number of cells and experimental animals required to produce equivalent data by between 4 and 40 fold. The protocol is designed to normalize results on a per cell basis, rather than to an internal control. This enables detection of overall reductions in protein levels that can be overlooked if data are normalized to an internal control. The importance of normalizing on a per cell basis was described for the analysis of gene expression data⁸, and the same principle applies to quantifying proteins by Western blot. This optimized protocol should be useful for anyone needing to analyze small numbers of cells.

Protokół

All procedures must be performed in accordance with institutional animal use and care guidelines. The procedure was developed for the analysis of murine hematopoietic stem and progenitor cells (HSCs and HPs), but can be adapted for the analysis of other cell populations.

1. Flow Cytometry Isolation of Murine HSCs and HPs

1. Harvest murine bone marrow cells as described in the literature⁶.
   NOTE: In data presented in Figure 1 and Figure 2, bone marrow was harvested from C57BL/6J mice.
2. Perform lineage depletion of bone marrow cells using a mouse lineage depletion kit, following the manufacturer's instructions.
3. Incubate the cells with antibodies against the cell surface markers of interest as described⁷. In the experiments shown in Figure 1 and Figure 2, antibodies to Sca-1, Kit, Flt3, and lineage (Lin) markers Mac1, Gr1, CD3e, B220, and Ter119 are used.
4. Sort 2,000 - 20,000 Lin^- Sca1⁺Kit⁺ Flt3^- cells (HSCs) or Lin^-Sca1^-Kit⁺ cells (HPs) into 1.5 mL Eppendorf tubes containing 0.1 mL of phosphate buffered saline (PBS) with 2% fetal bovine serum (FBS).
   NOTE: For the data shown in Figure 1 and Figure 2, cells were sorted using a Flow Cytometer with a 70 µM nozzle. The maximum collection volume is 1.4 mL.

2. Sample Preparation

NOTE: This step is critical. Process the sample very carefully. When removing the supernatant, be very careful not to disturb the cell pellet.

1. Centrifuge the 1.5 mL tubes containing sorted cells in a swinging bucket rotor at 4 °C, 500 x g, for 5 min. Remove all but approximately 100 µL of the supernatant from the cell pellet very gently using a pipette and a 20 µL pipet tip. Do not disturb the pellet.
   1. If the sample contains fewer than 5,000 cells, and the total volume of the sorted sample is less than 0.2 mL, transfer the cells first to low binding 0.2 mL PCR tubes before centrifugation.
   2. If sample contains fewer than 5,000 cells and volume is more than 0.2 mL, spin the cells down with a centrifuge at 4 °C, 500 x g, for 5 min, and remove all but 200 µL of the supernatant. Re-suspend the pelleted cells in the remaining 200 µL of supernatant, then transfer the cells to the 0.2 mL PCR tubes and spin down again. Remove all but 20 - 30 µL from the pellet after the second spin and re-suspend cells.
2. Resuspend the cells in the supernatant left in the tube. If necessary,add additional PBS in 2% FBS/PBS (you can also use 2% bovine serum albumin (BSA)/PBS, or PBS alone) to obtain a concentration 5 x10⁴ to 5 x 10⁵ cells/mL.
   NOTE: Use the cell counts collected by cytometry to estimate the volume used to suspend cells.
3. Use 2 - 5 µL of the re-suspended cells to determine an accurate cell concentration using an automated cell counter (following the manufacturer's instructions) or a hemocytometer⁹.
4. Transfer a volume of re-suspended cells containing the desired number of cells to new 1.5 mL tubes. For the experiment in Figure 1, 2,000, 1,000, or 500 HSCs or HPs were transferred. The desired number of cells depends on the strength of the signal obtained by Western blot, and is determined empirically. Perform the transfer using a 20 µL or 100 µL Eppendorf pipette tip.
5. Centrifuge the cells in a swinging bucket rotor at 4 °C, 500 x g, for 5 min.
6. To generate 100x stock solutions, dissolve proteasome and phosphatase inhibitors in DMSO according to the manufacturer's instructions.
7. Prepare 2x Laemmli sample buffer by diluting the 4x Laemmli sample buffer provided by the manufacturer with an equivalent amount of distilled water. Add an appropriate amount of the 100x stock of proteasome and phosphatase inhibitors to obtain a final concentration of 2x inhibitors in 2x Laemmli sample buffer.
   NOTE: The 2x Laemmli sample buffer can be made in advance, but the proteasome and phosphatase inhibitors should be added immediately before adding the 2x Laemmli sample buffer (plus inhibitors) to the cell pellet to lyse the cells, as described in the following step.
8. Carefully remove a portion of the supernatant from the cell pellet, and to the supernatant remaining in the tube with the cell pellet, add an equal volume of 2x Laemmli sample buffer plus proteasome and phosphatase inhibitors to achieve a final concentration of 500 cells/µL in 1x Laemmli sample buffer.
   NOTE: For example, if you transfer 2,000 cells in a total volume of 20 µL to a tube in Step 2.5, after centrifuging the cells (Step 2.6) you should remove 18 µL of the supernatant from the pellet, and to the 2 µL of supernatant that is remaining add an equal volume (2 µL) of 2x Laemmli sample buffer to achieve a final concentration of 500 cells/µL in 1x Laemmli sample buffer.
9. Resuspend the pellet to generate the lysate as soon as possible. At this point, the samples can be immediately electrophoresed through SDS-PAGE gels, or can be snap frozen in liquid N₂ and stored at -80 °C for future use.

3. Electrophoresis

1. Heat the lysates in a heat block at 95 °C or in boiling water for 5 min.
2. Electrophorese 1 - 40 µL of the lysates (containing from 500 up to 20,000 cell equivalents) through SDS-PAGE gels at 100V using standard protocols¹⁰.
   NOTE: The volume of lysate loaded into the gel is determined by the number of cells needed to generate the desirable signal, and will depend on the abundance of the protein of interest, and the quality of the antibody. The data in Figure 1 were obtained with 2,000, 1,000, and 500 cell equivalents of lysate. The width of the well should be in the range of 3 mm to 1.5 mm. 1.5 mm teeth can be cut from a commercially available comb with wider teeth using scissors.

4. Transfer and Block

NOTE: Perform a Western blot following standard protocols¹¹. The steps are briefly outlined here:

1. Pretreat a polyvinylidene difluoride (PVDF) membrane with methanol for 10 s, then wash the membrane briefly with distilled water.
2. Transfer the protein from the SDS-PAGE gel to the membrane following the instructions in the manual provided by the manufacturer of the transfer apparatus.
3. Following transfer, block the membrane with 2 mL of 5% bovine serum albumin (BSA) in PBST (PBS with 0.1% Tween) overnight at 4 °C following standard protocols¹¹.

5. Antibody Labeling

1. Incubate the membrane with antibodies on a shaker overnight at 4 °C using antibody dilutions recommended by the vendor.
   NOTE: In the Table of Materials, antibodies, which we have validated for HSCs and HPs and the corresponding antibody dilutions, are shown. Perform the antibody labeling using standard procedures⁸.

6. Detection

1. Wash the membrane 3 times, 5 min for each wash in PBST at room temperature.
2. Incubate the membrane with 2 mL of enhanced chemiluminescence buffer for 1 min at room temperature. Detect the signals using an imaging system following the manufacturer's instructions, or autoradiography film and film processor.

Wyniki

Representative results from 500 - 2,000 purified HSCs and HPs are shown in Figure 1 and Figure 2. The β-actin signal in Figure 1 can be detected from as few as 500 HSCs and HPs purified from the bone marrow of one mouse. Note that loading the lysates into 1.5 mm wells produced a much stronger signal from 500 HPs than loading into 3.0 mm wells. Figure 2 is a Western ...

Dyskusje

Western Blotting is a common technique for detecting specific proteins and the activation of signaling pathways in tissues or cells. By introducing small adjustments to a commonly used procedure, we were able to routinely detect 15 different proteins (Table of Materials) in 15,000 HSCs, and in some cases in as few as 500 HSCs. The most critical steps in this protocol are: 1) accurately counting the cells, 2) minimizing the number of transfers between tubes, and 3) lysing the cells directly with Laemmli s...

Materiały

Name	Company	Catalog Number	Comments
sodium dodecyl sulfate (SDS)	Fisher Scientific	BP166-500	SDS-PAGE
TEMED	Fisher Scientific	BP150-20	SDS-PAGE
30% Acrylamidel Bis solution	Bio-Rad	1610158	SDS-PAGE
1.5M Tris-HCl pH8.8	TEKNOVA	T1588	SDS-PAGE
1.0M Tris-HCl pH6.8	TEKNOVA	T1068	SDS-PAGE
Ammonium Persulfate	Fisher Scientific	BP179-25	SDS-PAGE
mini-protean 3 comb	Bio-Rad	1653360	SDS-PAGE
Mini-PROTEAN Tetra Cell	Bio-Rad	1658005EDU	SDS-PAGE
Transfer System	Bio-Rad	1704155EDU	transfer
PowerPac HC Power Supply	Bio-Rad	1645052EDU	electrophoresis
Imaging System	Bio-Rad	1708195	signal detection
4x Laemmli Sample Buffer	Bio-Rad	1610747EDU	sample preparation
10x Tris/Glycine/SDS Electrophoresis Buffer	Bio-Rad	1610732EDU	electrophoresis
2-Mercaptoethanol	Bio-Rad	1610710EDU	sample preparation
RTA Mini PVDF Transfer Kit, for 40 blots	Bio-Rad	1704272	transfer
Protease Inhibitor Cocktail	Sigma	11697498001	sample preparation
Phosphatase Inhibitor Cocktailrs	Gold Biotechnology	GB-450-1	sample preparation
EIF4G	Cell signaling	2469	WB antibody, validated in 1000 cells antibody concentration: 1:1000 reference(figure): Fig2
p-Rps6	Cell signaling	4858	WB antibody,validated in 1000 cells antibody concentration: 1:1000 reference(figure): Fig2
actin(HRP conjugated)	abcam	ab49900	WB antibody,validated in 500 cells antibody concentration: 1:5000 reference(figure): Fig1, Fig2
LC-3	Abcam	ab48394	WB antibody,validated in 15000 cells antibody concentration: 1:1000 reference(figure): ref5 (Fig5)
S6	Cell Signaling	2317	WB antibody,validated in 15000 cells antibody concentration: 1:1000 reference(figure): ref5 (Fig4)
4EBP1	Cell Signaling	9644	WB antibody, validated in 15000 cells antibody concentration: 1:1000 reference(figure): ref5 (Fig4)
p-4EBP1	Cell Signaling	2855	WB antibody,validated in 15000 cells antibody concentration: 1:1000 reference(figure): ref5 (Fig4)
TSC2	Cell Signaling	4308	WB antibody,validated in 15000 cells antibody concentration: 1:1000 reference(figure): ref5 (Fig4)
HSP90	Cell Signaling	4877	WB antibody,validated in 15000 cells antibody concentration: 1:1000 reference(figure): ref5 (Fig5)
PTEN	Cell Signaling	9188	WB antibody,validated in 15000 cells antibody concentration: 1:1000 reference(figure): ref5 (FigS1)
mTOR	Cell Signaling	2983	WB antibody,validated in 15000 cells antibody concentration: 1:1000 reference(figure): ref5 (FigS1)
p-mTOR	Cell Signaling	5536	WB antibody,validated in 15000 cells antibody concentration: 1:1000 reference(figure): ref5 (FigS1)
PP2AB	Cell Signaling	4953	WB antibody,validated in 15000 cells antibody concentration: 1:1000 reference(figure): ref5 (FigS1)
p-AMPKa	Cell Signaling	2535	WB antibody,validated in 15000 cells antibody concentration: 1:1000 reference(figure): ref5 (FigS1)
actin	Santa Cruz	sc-47778	WB antibody,validated in 15000 cells antibody concentration: 1:3000 reference(figure): ref5 (Fig4)
lineage depletion kit (mouse)	Miltenyi Biotec	130-090-858	hematopoietic stem and progenitor cells purification
LS columns	Miltenyi Biotec	130-041-305	hematopoietic stem and progenitor cells purification
SCF	PeproTech	250-03	phosphorylation stimulation
APC-Cy7 c-kit antibody	ThermoFisher	A15423	cell sorting
anti-B220	ThermoFisher	48-0452-82	cell sorting
anti-CD3e	ThermoFisher	48-0031-82	cell sorting
anti-Mac1	ThermoFisher	48-0112-82	cell sorting
anti-Gr1	ThermoFisher	48-5931-82	cell sorting
anti-Ter119	ThermoFisher	48-5921-82	cell sorting
Spacer Plates with 1.0 mm Integrated Spacers	Bio-Rad	1653311	SDS-PAGE
BSA	Research Products International	A30075	membrane blocking
serum	Atlanta Biologicals	A12450	medium supplement
cell counter	Invitrogen	AMQAF1000	cell counting
hemocytometer	ThermoFisher	S17040	cell counting
flim	Denville Scientific	E3012	signal detection
medical film processor	Konica	SRC-101A	signal detection
Supersignal West Femto Maximum Sensitivity Substrate	Therom Scientific	34096	signal detection
Allegra X-15R Centrifuge (Rotor SX4750A)	Beckman Coulter	X-15R	sample preparation
BLUEstain protein ladder	Gold Biotechnology	P007-500	electrophoresis
cell sorter	BD	FACSAria II	sample preparation
Incublock	Denville Scientific	I0510	electrophoresis