Can I Centrifuge Samples in Buffer Laemmli Again

J Vis Exp. 2018; (138): 56855.

A Western Blotting Protocol for Pocket-size Numbers of Hematopoietic Stem Cells

Xiongwei Cai

¹Partitioning of Experimental Hematology, Cincinnati Children's Hospital Medical Eye

²Department of Cell and Developmental Biology, Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania

ⁱⁱⁱDepartment of Obstetrics and Gynecology, Southwest Hospital, Third Military Medical University

Yi Zheng

ⁱDivision of Experimental Hematology, Cincinnati Children'southward Hospital Medical Eye

Nancy A. Speck

^twoDepartment of Cell and Developmental Biology, Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania

Abstract

Hematopoietic stalk cells (HSCs) are rare cells, with the mouse os marrow containing but ~25,000 phenotypic long term repopulating HSCs. A Western blotting protocol was optimized and suitable for the assay of small numbers of HSCs (500 - fifteen,000 cells). Phenotypic HSCs were purified, accurately counted, and direct 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 - five,000 HSCs can be routinely analyzed, and in some cases information tin can be obtained from as few as 500 cells, compared to the twenty,000 to 40,000 cells reported in nigh publications. This protocol should be by and large applicable to other hematopoietic cells, and enables the routine analysis of small numbers of cells using standard laboratory procedures.

Keywords: Biochemistry, Issue 138, Western blotting, hematopoietic stem cells, pocket-sized cell numbers, SDS-Page, fauna testing reduce, bone marrow

Introduction

Hematopoietic stem cells (HSCs) are self-renewing cells that tin requite rise to all blood lineages. They are relatively rare cells in the bone marrow, rendering biochemical analyses hard. Approaches suitable for analyzing rare cells, such as menstruation 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 antibiotic access, and non all cell surface epitopes survive these procedures1,2. In add-on, antibodies that discriminate betwixt different poly peptide isoforms or cleavage products are not oftentimes available for menstruation cytometry, and therefore investigators still rely on Western blots for sure types of analyses.

Western blot analysis of cell lysates is a routine procedure in near laboratories. Cells tin be purified under native weather that preserve the epitopes of prison cell surface molecules, and cell lysates tin can subsequently be prepared and analyzed. However, the analysis of proteins in rare chief cell populations past Western blot can require euthanizing large numbers of animals to obtain plenty cells. Past making small adjustments to several steps, a conventional Western blotting protocol was able to detect proteins in relatively modest numbers of HSCs (500 - 15,000, depending on the protein of interest). The adjustments include accurately counting the cells, carefully treatment the prison cell pellet, reducing transfers of cells between tubes to minimize cell loss, and lysing a divers number of cells with a concentrated loading buffer containing proteasome and phosphatase inhibitors. Many published reports include Western blots obtained with xx,000 or more HSCs3,4,5,6,7; 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 ground, rather than to an internal command. This enables detection of overall reductions in protein levels that can be overlooked if information are normalized to an internal control. The importance of normalizing on a per cell ground was described for the analysis of cistron expression data8, and the aforementioned principle applies to quantifying proteins past Western absorb. This optimized protocol should be useful for anyone needing to analyze minor numbers of cells.

Protocol

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

i. Menstruation Cytometry Isolation of Murine HSCs and HPs

Harvest murine os marrow cells every bit described in the literature6. NOTE: In information presented in Effigy 1 and Figure 2, bone marrow was harvested from C57BL/6J mice.
Perform lineage depletion of bone marrow cells using a mouse lineage depletion kit, following the manufacturer's instructions.
Incubate the cells with antibodies against the cell surface markers of interest as described7. In the experiments shown in Figure 1 and Figure two, antibodies to Sca-1, Kit, Flt3, and lineage (Lin) markers Mac1, Gr1, CD3e, B220, and Ter119 are used.
Sort 2,000 - xx,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 Effigy 1 and Effigy 2, cells were sorted using a Flow Cytometer with a lxx µM nozzle. The maximum collection book is 1.4 mL.

2. Sample Grooming

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

Centrifuge the i.five mL tubes containing sorted cells in a swinging bucket rotor at iv °C, 500 x one thousand, 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 non disturb the pellet.
1. If the sample contains fewer than v,000 cells, and the total volume of the sorted sample is less than 0.2 mL, transfer the cells start to low binding 0.2 mL PCR tubes before centrifugation.
2. If sample contains fewer than v,000 cells and volume is more than than 0.ii mL, spin the cells down with a centrifuge at 4 °C, 500 10 yard, for v min, and remove all but 200 µL of the supernatant. Re-suspend the pelleted cells in the remaining 200 µL of supernatant, and then transfer the cells to the 0.two mL PCR tubes and spin down once more. Remove all but 20 - 30 µL from the pellet afterwards the 2d spin and re-suspend cells.
Resuspend the cells in the supernatant left in the tube. If necessary,add together additional PBS in two% FBS/PBS (y'all can also use 2% bovine serum albumin (BSA)/PBS, or PBS alone) to obtain a concentration 5 x10⁴ to v x 10^five cells/mL. Note: Utilize the cell counts collected by cytometry to estimate the volume used to suspend cells.
Use ii - 5 µL of the re-suspended cells to determine an accurate cell concentration using an automated prison cell counter (following the manufacturer's instructions) or a hemocytometer9.
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 force of the signal obtained by Western blot, and is adamant empirically. Perform the transfer using a 20 µL or 100 µL Eppendorf pipette tip.
Centrifuge the cells in a swinging saucepan rotor at iv °C, 500 ten chiliad, for 5 min.
To generate 100x stock solutions, dissolve proteasome and phosphatase inhibitors in DMSO according to the manufacturer's instructions.
Fix 2x Laemmli sample buffer by diluting the 4x Laemmli sample buffer provided by the manufacturer with an equivalent corporeality of distilled water. Add together an advisable amount of the 100x stock of proteasome and phosphatase inhibitors to obtain a concluding concentration of 2x inhibitors in 2x Laemmli sample buffer. Notation: The 2x Laemmli sample buffer can be made in advance, merely the proteasome and phosphatase inhibitors should be added immediately before calculation the 2x Laemmli sample buffer (plus inhibitors) to the cell pellet to lyse the cells, as described in the following step.
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 book 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 lot transfer ii,000 cells in a full volume of xx µL to a tube in Footstep 2.v, later on centrifuging the cells (Step ii.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 book (2 µL) of 2x Laemmli sample buffer to achieve a terminal concentration of 500 cells/µL in 1x Laemmli sample buffer.
Resuspend the pellet to generate the lysate equally presently as possible. At this point, the samples can be immediately electrophoresed through SDS-Folio gels, or can exist snap frozen in liquid N₂ and stored at -80 °C for future employ.

3. Electrophoresis

Heat the lysates in a heat block at 95 °C or in humid water for 5 min.
Electrophorese 1 - 40 µL of the lysates (containing from 500 up to 20,000 jail cell equivalents) through SDS-Folio gels at 100V using standard protocols10. Annotation: The volume of lysate loaded into the gel is adamant 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 two,000, one,000, and 500 cell equivalents of lysate. The width of the well should be in the range of 3 mm to one.5 mm. one.five mm teeth can be cut from a commercially available rummage with wider teeth using scissors.

4. Transfer and Cake

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

Pretreat a polyvinylidene difluoride (PVDF) membrane with methanol for 10 s, and so wash the membrane briefly with distilled h2o.
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.
Post-obit transfer, cake 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.

five. Antibody Labeling

Incubate the membrane with antibodies on a shaker overnight at 4 °C using antibody dilutions recommended past the vendor. NOTE: In the Tabular array of Materials, antibodies, which nosotros take validated for HSCs and HPs and the corresponding antibody dilutions, are shown. Perform the antibiotic labeling using standard procedures8.

half dozen. Detection

Wash the membrane three times, 5 min for each launder in PBST at room temperature.
Incubate the membrane with 2 mL of enhanced chemiluminescence buffer for one min at room temperature. Detect the signals using an imaging system following the manufacturer'due south instructions, or autoradiography motion picture and motion picture processor.

Representative Results

Representative results from 500 - 2,000 purified HSCs and HPs are shown in Figure 1 and Figure 2. The β-actin point in Figure 1 can be detected from equally few every bit 500 HSCs and HPs purified from the bone marrow of one mouse. Note that loading the lysates into one.5 mm wells produced a much stronger bespeak from 500 HPs than loading into 3.0 mm wells. Figure 2 is a Western absorb of EIF4G and the phosphorylation of Rps6 (p-Rps6), both of which are involved in the regulation of protein translation12, in HSCs, and in HPs with and without stimulation by stem prison cell factor (SCF) in vitro.

Figure ane: Western Blot for β-actin performed with lysates from murine HSCs and HPs. HSCs were sorted from lineage depleted murine bone marrow cells as Lin^-Sca1⁺Kit⁺ Flt3^- cells, and HPs were Lin^-Sca1^-Kit⁺. Lysates prepared from different numbers of cells were electrophoresed through a 12% SDS-PAGE gel prepared using mini glass plates with 1 mm spacers. The absorb was probed with antibody to β-actin. The absorb shows the comparative β-actin signals from 500 to 2,000 HPs when the lysates were loaded into 1.5 mm wells (lanes four - 6) as compared to 3 mm wells (lanes i - 2). Lysates from ii,000 to 500 cells were loaded onto lanes seven to nine. Yard, molecular weight markers. Please click here to view a larger version of this effigy.

Effigy 2: Western Absorb assay of freshly isolated HSCs, and HPs stimulated in vitro with the cytokine Stem Jail cell Cistron (SCF). HPs were purified by fluorescence activated cell sorting (FACS), centrifuged, resuspended in two% FBS/PBS, counted, then carve up into two tubes. The HPs in the start tube were stimulated with SCF (10 ng/mL) for 5 min at 37 °C (+SCF), and HPs in the second tube were cultured for 5 min in the absence of SCF (-SCF). The cells were and so centrifuged and the cell pellet lysed with Laemmli sample buffer. HSCs were purified and directly lysed with Laemmli sample buffer without culturing. The lysates were loaded into 1.v mm wells and electrophoresed through 12% SDS-PAGE gels. The blot was developed with antibodies to elongation initiating factor 4G (EIF4G), β-actin, and phosphorylated small ribosome subunit S6 (p-Rps6). Please click here to view a larger version of this figure.

Discussion

Western Blotting is a common technique for detecting specific proteins and the activation of signaling pathways in tissues or cells. By introducing minor adjustments to a usually used procedure, nosotros were able to routinely detect fifteen different proteins (Table of Materials) in 15,000 HSCs, and in some cases in equally few as 500 HSCs. The most critical steps in this protocol are: 1) accurately counting the cells, 2) minimizing the number of transfers betwixt tubes, and 3) lysing the cells directly with Laemmli sample buffer. When lysing the cell pellet with Laemmli sample buffer, nosotros found that rather than removing all of the supernatant from the cell pellet and re-suspending it in Laemmli Sample Buffer, if we instead left a small book of supernatant in the tube and added an equivalent book of 2x Laemmli Sample Buffer, nosotros could avoid cell loss. Centrifuging the cells in a swinging bucket rotor also decreased the risk of prison cell loss. Reducing the width of the well by trimming the comb teeth improved the sensitivity of detection.

With these simple adjustments to the process, we were able to obtain reproducible results equivalent to those in published papers that had used 10 - 20 times more cells3,iv,v,6,vii. Further, the blots can exist stripped for re-blotting following standard procedures13, increasing the amount of data that can be obtained from a small number of cells. The power to detect proteins of interest volition be limited by the quality of the antibody and the protein abundance. This modified technique should profoundly reduce the number of animals necessary to obtain protein information from rare cell populations.

Disclosures

The authors take no conflict of interests to declare.

Acknowledgments

National Institutes of Health grant R01 CA149976 (North.A.S) supported this piece of work.

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Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6231698/